Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase. Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.


Phosphorus-31 nuclear magnetic resonance study of D-serine dehydratase: pryridoxal phosphate binding site. The pyridoxal phosphate dependent enzyme D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In the native enzyme, the pyridoxal phosphate 31P chemical shift is pH dependent with pKa = 6.4, indicating exposure of the phosphate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-pyridoxal phosphate complex. This transaldimination complex is fixed to the enzyme via the phosphate group of the cofactor as the dianion, independent of pH. At pH 6.6 the dissociation constant KD for isoserine determined by NMR is 0.43 mM. Reconstitution of the apoenzyme with pyridoxal phosphate monomethyl ester produces an inactive enzyme. NMR and fluorescence measurements show that this enzyme does not form the transaldimination complex, indicating that the fixation of the dianionic phosphate (probably via a salt bridge with an arginine residue) observed in the native enzyme is required for the transaldimination step of the catalytic mechanism.


Near-heme histidine residues of deoxy- and oxymyoglobins. Proton NMR titration curves of the histidine Cepsilon-H resonances of the deoxy and oxy forms of human, horse, and sperm whale myoglobins (Mb) were determined and compared with the results for the met and azide forms. One extra titrating resonance (H-8) was observed for each deoxy-Mb compared with the corresponding met-Mb, and a further extra resonance (H-9) was observed for the oxy-Mb form. These resonances correspond to the two additional resonances previously described for azide-Mb [Hayes, M., Hagenmaier, H., & Cohen, J. S. (1975) J. Biol. Chem. 250, 7461--7472]. This new evidence prompts us to reassign these resonances to the near-heme histidine residues.


Interpretation of nuclear magnetic resonance spectra for Lactobacillus casei dihydrofolate reductase based on the X-ray structure of the enzyme-methotrexate-NADPH complex. The three-dimensional molecular structure of Lactobacillus casei dihydrofolate reductase complexed with NADPH and methotrexate has been used to interpret published magnetic resonance spectra for this enzyme. Proton resonances from histidine residues and 19F resonances from fluorine-labeled fluorotyrosine and fluorotryptophan dihydrofolate reductase have been assigned in several cases to specific amino acids in the primary sequence. Furthermore, the 31P signals from the pyrophosphate moiety of bound NADPH have been assigned and the large upfield shift for 13C-labeled (at the carboxamide carbon) NADP+ upon binding to the reductase has been explained in terms of desolvation effects.


Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome. Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.


Transient spectra of intermediates in the photolytic sequence of octopus rhodopsin. The intermediate photolytic sequence of octopus rhodopsin was studied at different temperatures and different pH values by means of a flash photolysis-rapid scan spectrophotometry near physiological temperature. The first photoproduct in the photolysis of rhodopsin was lumirhodopsin. Transformation of lumirhodopsin leads to mesorhodopsin took place independently of the pH of the solution. Mesorhodopsin was transformed to acid metarhodopsin in acid solution. In alkaline solution, mesorhodopsin was transformed to transient acid metarhodopsin whose absorption spectrum was similar to acid metarhodopsin. Transient acid metarhodopsin was then transformed to alkaline metarhodopsin reaching a tautomeric equilibrium which was determined by the pH of the solution.


Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate. The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.


Kinetics of sulfate uptake by yeast.

Kinetics of sulfate uptake by yeast. Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H+ and an efflux of 1 K+ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but Km is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.


Evidence against a MgATP-dependent proton pump in rat-liver lysosomes. 1. The effect of MgATP has been studied on the accumulation of the lipid-soluble anion thiocyanate, the accumulation of the lipid-soluble base methylamine, and the fluorescence of bound anilinonaphthalene sulphonate in rat-liver lysosomes. The lysosomes used were isolated from the livers of rats pretreated with Triton WR 1339. 2. The accumulation of thiocyanate is stimulated by the addition of valinomycin in the presence of K+ but not by the addition of MgATP. 3. The fluorescence of anilinonaphthalene sulphonate bound to lysosomes is enhanced by valinomycin in the presence of K+, the extent of the enhancement being dependent on the concentration of K+. In contrast, MgATP has no effect on the fluorescence. 4. The intralysosomal pH, as estimated from the distribution of methylamine, is not affected by the addition of MgATP in media with or without K+, Na+ or HCO3-. 5. These data strongly suggest that there is no MgATP-dependent proton pump in rat-liver lysosomes.


An improved method for the isolation and assay of the acid lipase from human liver. An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.


The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride. The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.


Nuclear magnetic resonance study of side-chain conformation of phenylalanine residue in [Met5]-enkephalin: solvent, pH, and temperature dependence. [Met5]-Enkephalin and N-acetylphenylalanine methylamide containing (2S,3S)-[2,3-2H2]Phe were synthesized 270 MHz 1H NMR spectra of the normal and selectively deuterated species were analysed. The lower-field and higher-field beta-proton signals of the Phe4 residue of [Met5]-enkephalin were unambiguously assigned to the pro-S and pro-R protons, respectively. The same assignments apply to N-acetylphenylalanine methylamide in polar organic solvents and in 2H2O, but the alternative assignments apply in C2HCl3. For [Met5]-enkephalin, the vicinal spin coupling constants 3JalphabetaS and 3 JalphabetaR and the rotamer populations around the Calpha-Cbeta bond were determined in a variety of solvents. From the pH and temperature dependences of rotamer populations of [Met5]-enkephalin, the side-chain conformation of the Phe residue in 2H2O solution was found to be considerably different from that in (C2H3)2SO solution. Rotamer populations of the Phe4 residue of [Met5]-enkephalin in organic solvents depend on solvent polarity. As compared with the reference model molecule of N-acetylphenylalanine methylamide, the rotamer populations of Phe4 of [Met5]-enkephalin are affected possibly by steric repulsion with other residues; the rotamer I is primarily favored but the rotamer II is appreciably destabilized in weakly polar solvents.


Isolation and partial characterization of intermediate filament protein (skeletin) from cow heart Purkinje fibres. The intermediate filament protein skeletin from cow heart Purkinje fibres was purified to homogeneity by a selective extraction procedure and gel chromatography in the presence of sodium dodecyl sulphate. Monospecific antibodies were obtained by immunisation of rabbits with the sodium dodecyl sulphate-skeletin complex, and rocket electrophoresis made it possible to quantify the concentration of protein. The skeletin monomer has a molecular weight of 55 000. Amino acid analysis revealed that skeletin has a high content of glutamic acid, aspartic acid, alanine and leucine, together constituting more than 50% of the molecule. The isoelectric point is determined as 6.35. Skeletin is insoluble at pH 4--6 in the absence of detergent and shows increasing solubility at higher and lower pH. The biochemical characteristics are discussed in relation to the cytoskeletal function of the filaments. Comparison with intermediate-sized filament protein of other tissues show certain important similarities suggesting that the filaments may share a common evolutionary ancestry.


Separation of soluble adenylate and guanylate cyclases from the mature rat testis. The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.


Non-H-2 antigens can induce high GVH mortality in adult recipients of normal cells. It is demonstrated that, under experimental conditions resembling those used for bone marrow grafting in man, disparity for minor histocompatibility antigens alone (i.e., antigens coded for by genes not included in the major histocompatibility complex) is in fact sufficient for the induction of severe lethal graft-versus-host disease in adult (DBA/2 X B10.D2)F1 recipients of normal B10.D2 myeloid and lymphoid cells.


The analysis of terbutaline in biological fluids by gas chromatography electron impace mass spectrometry. A highly sensitive and specific assay is described for the bronchodilator drug terbutaline in human plasma and urine, based on single ion monitoring gas chromatography mass spectrometry and employing a homologue of the drug as internal standard. Recovery of terbutaline and internal standard into ethyl acetate is effected at pH 9.8, while back-extraction into dilute acid serves to purify the initial extract. Following preparation of O-TMS, N-TFA derivatives, the drug and its homologue are detected by selected ion monitoring of their common base ion at m/e 355. The limit of detection of terbutaline by this procedure is 0.3 ng ml-1 from a 4 ml sample of plasma.


Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose. Trichoderma can be cultured in stirred-tank fermentors on high (8%) cellulose concentrations without increasing the salt concentration of the medium when NH4OH is used to control pH and as a nitrogen source. Approximately 90% of the ammonia consumed by the organism can be added as NH4OH. The advantage of using high concentrations of cellulose is that culture filtrates with greater cellulase activity are obtained. The advantage of a low salts medium is that unwanted solutes in the final enzyme preparation are reduced. The appearance of cellulase in the medium occurs later than net ammonia uptake so that only 20% of the final amount of cellulase has appeared when 80% of the maximum amount of ammonia has been consumed.


Immobilization of aminoacylase by adsorption to tannin immobilized on aminohexyl cellulose. The immobilization of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by using tannin immobilized on aminohexyl cellulose. The most active immobilized aminoacylase was obtained when aminoacylase was adsorbed to the immobilized tannin in a weak alkaline medium containing sodium chloride and n-butanol at 37 degrees C. The activity of the immobilized tannin-aminoacylase complex per unit volume was five times higher than that of the DEAE-Sephadex-aminoacylase complex used for industrial production of L-amino acids in our plants. The half-life of the immobilized tannin-aminoacylase complex was 20 days under continuous operation at a high concentration of substrate; on the contrary, that of the DEAE-Sephadex-aminoacylase complex was 0.5 days.


[General characteristics and comparative evaluation of the radioprotective properties of aryl alkyl amine adrenomimetics in experiments on mice]. The radioprotective effect (RPE) of some arylalkylamines (AAAs) was studied in experiments on mice. Mesaton and its close analogues were injected subcutaneously 15 minutes prior to irradiation at a dose of 800 rad. The protective effect is exerted by AAAs in low doses (25--50 mumole/kg), the compounds show stable and high RPE (80--80% survival, dose reduction factor being 1.3--1.4) and low toxicity (LD50 = 4--8 mumole/kg). AAAs studied are not less effective than aminothiols. Their pharmacological spectrum--K = LD50/ED50 (200--500) is superior to that of known aminothiols and indolylalkylamines.


[Phenomenon of parental resistance and its genetic regulation]. Lymphocytes of mice F1 (CBA X M523) and F1 (A X M523) transplanted to 1000 R irradiated CBA or A mice responded to the test antigens--SRBC or S. typhi Vi-antigen--by formation of 100--1000 times less antibody forming cells than in syngeneic recipients. An intermediate result is achieved when the lymphoid cells are transplanted to the irradiated M523 mice. Lymphocytes of mice F1 (A X CBA), F1 (CBA X C57Bl/6), or F1 (A X A.CA) developed a similar immune response in the irradiated syngeneic mice and in both parental lines. The ability of parental line M523 to respond to SRBC was the same as in the other lines studied when examined in situ or in adoptive transfer experiments. The stem hemopoietic cells of mice F1 (CBA X M523) develop in the spleen of CBA mice 2--2.5 times less hemopoietic colonies than in the spleen of syngeneic animals. A conclusion was drawn that mutation M523 in CBA mice inhibited the proliferation and differentiation of hemopoietic and lymphoid cells in the irradiated nonsyngeneic recipients.


[Liver reactivity to glucocorticoids in the process of chemical hepatic carcinogenesis]. The rat hepatocytes during chemical carcinogenesis (3'-MDAB), as well as the cells of chemically-induced primary rat hepatomas preserved their response to partial hepatectomy by stimulation of the 3H-thymidine incorporation into DNA; as in the normal liver this process is inhibited by dexamethason. No impairment in the inducibility of tyrosine aminotranspherase (EC.2.6.1.5) by the hormone was observed, whereas the hormonal induction of tryptophane pyrrolase (EC.1.13.11.11) in primary hepatomas was lost completely. The problem of the adequacy of the model of chemical carcinogenesis of the organ without consideration of the cell populations heterogeneity is discussed.


[Activation of tumor tissue glycolysis by inorganic phosphate at low pH values]. It was shown that the activating effect of inorganic phosphorus on glucose utilization by the tumour tissue in vitro was retained at low pH of the incubation medium. The 0.15M Na2HPO4 in tris-buffer solutions infusion at the moment of cessation of the tumour selfacidity under glucose infusion led to further decrease of the tumour tissue pH. The tumour tissue pH values of about 4.5--4.6 were obtained. It is recommended to use the solutions with inorganic phosphorus for acidifying the tumour tissue in optimisation of the complex therapy schemes.


The role of the hypogastric nerve in bladder and urethral activity of the dog. 1. Stimulation of the hypogastric nerves increased the pressure in both the bladder and urethra of anaesthetized female dogs. 2. The responses were reduced but not abolished by the alpha-adrenoceptor antagonist phentolamine, whereas the beta-adrenoceptor antagonist propranolol was either without effect or increased the responses. Atropine, methysergide and hexamethonium were without effect. 3. Close arterial injection of phenylephrine increased and isoprenaline decreased urethral pressure but both produced only a slight increase in bladder pressure. 4. Hypogastric nerve stimulation reduced subsequent responses of the bladder and urethra to pelvic nerve stimulation or to close arterial injection of acetylcholine. Isoprenaline, but not phenylephrine, also had an inhibitory action and 5-hydroxytryptamine enhanced the responses. 5. In the presence of hexamethonium the inhibitory action of isoprenaline still occurred but 5-hydroxytryptamine no longer enhanced the responses, suggesting that 5-hydroxytryptamine acts on the ganglia and isoprenaline acts, at least partially, on smooth muscle. 6. These results suggest that the role of the hypogastric nerves may be to modify inputs to the bladder and urethra as well as to act directly on the smooth muscle.


beta-Adrenoceptor blocking drugs and isoprenaline: central effects on cardiovascular parameters. 1. The central hypotensive activity of (+)- and (-)-propranolol (100 microgram), pindolol (100 microgram) and isoprenaline (1 and 4 microgram) injected intracerebroventricularly (i.c.v.) was studied in rats anaesthetized with urethane and chloralose. Blood pressure, cardiac output and heart rate were measured; systolic stroke volume and peripheral vascular resistance were calculated. 2. (+)- and (-)-Propranolol and pindolol induced a fall of blood pressure but (+)-propranolol was less active. The heart rate was reduced more by (-)-propranolol than by (+)-propranolol or (-)-pindolol. The decrease of systolic stroke volume was greater for (-)-propranolol and pindolol than for (+)-propranolol. Peripheral vascular resistance was reduced to the same level but with different time courses, (-)-propranolol having a longer effect than (+)-propranolol and pindolol. 3. Isoprenaline induced a hypotensive effect, while cardiac output and heart rate increased; the systolic stroke volume remained stable but peripheral vascular resistance was significantly decreased. 4. These results suggest that different central regulatory centres are involved in the control of cardiac function and peripheral vascular tone.


Catecholamine synthesizing enzymes in brain stem and hypothalamus during the development of renovascular hypertension. The activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) have been measured in brain stem and hypothalamic nuclei during the development of renovascular hypertension. TH activity fell at 72 h in the posterior hypothalamic and peri- and paraventricular nuclei of the hypothalamus, but had returned to control levels by 7 days. PNMT activity was raised in the nucleus of the solitary tract at 7 days and was also elevated in the nucleus of the solitary tract, parahypoglossal nucleus, locus coeruleus and cerebellar cortex at 4 weeks. No change in PNMT was noted in hypothalamus. It appears from these results that both central noradrenergic and adrenergic pathways are involved in the development of this model of experimental hypertension.


Long-term effects of preganglionic nerve stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion. The effects of increased synaptic stimulation of sympathetic neurons on the tyrosine hydroxylase activity of these cells were studied. Seventy-two hours after unilateral stimulation of the preganglionic cervical sympathetic trunk at 10 Hz for 30 min tyrosine hydroxylase activity was 32% higher in stimulated than in control superior cervical ganglia. Stimulation at 10 Hz for only 10 min increased enzyme activity by 25% when measured 72 h later, while stimulation for 60 min increased activity by 73%. No further change in enzyme activity was found after 90 min of stimulation although electrophysiological recordings from the ganglion demonstrated that synaptic transmission was maintained throughout the period of stimulation. Ganglionic neurons also follow high frequency trains of stimuli when they are interrupted by silent periods. Stimulation with 40 Hz trains (250 msec on/500 msec off) for 30 min and 90 min produced a 50% and a 92% increase in tyrosine hydroxylase activity, respectively. Stimulation of ganglia with the same number of pulses administered either continuously or in trains produced the same size increase in enzyme activity. The relationship between preganglionic nerve activity and tyrosine hydroxylase activity may represent an adaption of sympathetic neurons to situations requiring increased transmitter release.


Regional development of norepinephrine, dopamine-beta-hydroxylase and tyrosine hydroxylase in the rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine. Neonatal rats were injected subcutaneously with 100 mg/kg 6-hydroxydopamine (6-OHDA), or vehicle, on postnatal days 1, 2 and 3. At several times thereafter, determinations of tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities, and norepinephrine (NE) concentration were made in the parietal cortex, cerebellum and pons-medulla in order to assess the extent of initial noradrenergic degeneration induced, and the rate of any ensuing regeneration. By the day following completion of the treatment (postnatal day 4), degeneration of noradrenergic terminals in the parietal cortex and cerebellum was very extensive, with NE levels and DBH activities reduced by more than 80%, and TOH activities reduced by 50%. In the parietal cortex noradrenergic degeneration remained virtually complete; and 9 and 70 days postnatal NE concentration and DBH and TOH activities were all decreased by more than 90--95%. In the cerebellum a progressive regeneration and apparent sprouting of NE fibers was observed. By postnatal day 9, NE, DBH and TOH in this tissue had all recovered to near control levels, and by day 70 these measures exceeded control levels by 95%, 115% and 50% respectively. In the pons-medulla, the initial effect of 6-OHDA on any of the measured parameters was negligible. By postnatal day 9 an increase in NE concentration was apparent, which increased further by day 70 to surpass the control level by 70%. At this same time DBH activity was increased by only 15% and TOH activity was unchanged. Separate analysis of the rostral half of the pons, which contains the locus coeruleus, revealed that on day 70 NE and DBH levels were increased much more substantially than in the whole pons-medulla, and TOH activity was also significantly elevated. This data indicates that the initial amount of degeneration induced by the 6-OHDA treatment is similar in both the parietal cortex and cerebellum, but regeneration proceeds only in the cerebellum. This suggests that noradrenergic fiber growth and regeneration in each target tissue is under independent regulation, possibly by the individual target neurons themselves.


Assessment of the effects of neonatal subcutaneous 6-hydroxydopamine on noradrenergic and dopaminergic innervation of the cerebral cortex. Female rats, treated at birth with 6-hydroxydopamine (3 x 100 mg/kg s.c. at 24 h intervals) or vehicle, were subjected at 112 days of age to unilateral electrolytic lesions of the locus coeruleus. Two weeks later regions of the telencephalon, both ipsi- and contralateral to the lesion, were simultaneously assayed for norepinephrine (NE) and dopamine (DA) content, and for tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities. In the vehicle-treated rats the lesion resulted in at least an 80% reduction of NE and DBH on the ipsilateral side, relative to the contralateral side. TOH was reduced to a similar extent only in the parietal cortex and hippocampus. In the prefrontal cortex and cingulate gyrus TOH was decreased by only 31% and 64% respectively; the remainder was interpreted to be associated with projections of the mesocortical dopamine system. From this data it was possible to calculate that the ratio of TOH to DA in dopaminergic terminals is about 10-fold greater than the ratio of TOH to NE in noradrenergic terminals. Neonatal 6-hydroxydopamine treatment resulted in practically total elimination of noradrenergic terminals throughout the telencephalon, and the locus coeruleus lesion had no additional effect. The drug treatment produced no significant change in DA content or in the TOH to DA ratio in the prefrontal cortex and cingulate gyrus, indicating complete sparing of the mesocortical DA projections.


[Comparative effects of antiepileptics on the alkaline phosphatase and gamma glutamyltransferase activities in plasma and leukocytes]. Alkaline phosphatase and gamma glutamyltransferase activities have been compared in plasma and leukocytes from presumably healthy subjects and from epileptic patients under treatment with different antiepileptic drugs. Plasma enzyme activities are always increased by antiepileptic treatment, leukocytic enzyme activities are increased only for some patients. No relation has been observed between the variations in any of the two enzyme activities and plasma level of anticonvulsant drugs.


Hemolysins of Edwardsiella tarda.

Hemolysins of Edwardsiella tarda. Isolates of Edwardsiella tarda from four sources produced nonfilterable hemolsin in trypticase soy broth. The cell-associated hemolysin was partially heat labile, destroyed by formalin and sensitive to treatment with trypsin. These characteristics, and the observation that Ca++ or Mg++ ions enhanced activity, suggest that a proteinaceous, enzymic component may be responsible for the hemolytic activity.


Cardiac muscarinic blocking and atropinic blocking effects of a tetramine disulfide and alpha-adrenoceptor blocking activity. Since many alpha-adrenoceptor blocking drugs were known to have muscarinic blocking activity, BHC, a newly synthesized tetramine disulfide with alpha-adrenoceptor blocking activity, and two of its analogs were tested for muscarinic blocking activity in the isolated guinea pig atrium and ileum. BHC had muscarinic blocking activity. Its potency was higher in the atrium than in the ileum. The cardiac muscarinic blocking potency of the analog that contained carbon atoms instead of sulfur atoms was similar to that of BHC. Lower concentrations of the drugs antagonized inotropic effects of acetylcholine competitively, but higher concentrations had a smaller effect than expected for competitive antagonism. In the atrium BHC competitively inhibited the muscarinic blocking effect of atropine. Its atropinic blocking potency was similar to the muscarinic blocking potency. Quinidine and gallamine also had muscarinic blocking and atropinic blocking effects in the atrium but were about 10 times less potent than BHC.


California encephalitis virus activity in mosquitoes and horses in southern Ontario, 1975. A study was undertaken in 1975 to determine California encephalitis virus activity in southern Ontario. Three thousand and sixty-one mosquitoes, primarily Aedes species, were divided into 104 pools and inoculated into suckling mice. Isolates of snowshoe hare virus were obtained from one pool each of Aedes fitchii and A. triseriatus mosquitoes collected in the Guelph area. Serological testing of horse sera revealed extensive virus activity in southern Ontario and indicated that horses may serve as excellent monitors for California encephalitis virus.


Enumeration of stressed cells of Escherichia coli. Cells of Escherichia coli K-12 were stressed by heating at 48 degrees C or by acid treatment at pH 4.2 for periods up to 1h. The addition of catalase to the selective medium increased the count of heat-stressed cells by 2.3-fold and acid-stressed cells by 4.8-fold. However, these values represented only a small percentage (3% for heat-stressed and 6% for acid-stressed cells respectively) of the population of injured but still viable cells. The addition of mannitol to the selective medium used to count acid-stressed cells did not increase the count. Whilst the presence of H2O2 in media may cause significant errors in the estimation of E. coli in certain situations these errors are unlikely to be significant in physiological studies of populations of cells injured by stress.


Response of cells to hyperthermia under acute and chronic hypoxic conditions. The lethal response of Chinese hamster cells heated to 42 degrees was determined following 0 to 30 hr culturing under hypoxic conditions. Oxygenated and acutely hypoxic cells were equally sensitive to hyperthermia; however, sensitivity increased with the time of culturing under hypoxic conditions prior to treatment. Three hr at 42 degrees resulted in a surviving fraction of approximately or equal to 0.1 under acute hypoxic conditions and less than 0.001 for cells cultured for 30 hr under oxygen-deprived conditions before the heat treatment. The increased sensitivity to hyperthermia was was due in part to a decrease in the pH of the medium which occurred as a result of cell metabolism; this could be reversed by increasing pH to 7.3 immediately prior to heat treatment. However, even under fully controlled pH conditions, prolonged oxygen deprivation increased hyperthermic cell killing by a factor of approximately or equal to 5. This effect was not reversed by returning the cells to normal oxygen tension prior to treatment. These data demonstrate that tumorlike microenvironmental conditions (reduced O2 tension and pH) substantially increase the sensitivity of cells to 42 degrees hyperthermia.


Evidence for several demethylase enzymes in the oxidation of dimethylnitrosamine and phenylmethylnitrosamine by rat liver fractions. The steady state kinetics and isotope effects were examined for demethylation of dimethylnitrosamine and phenylmethylnitrosamine, as well as their deuterated analogs, using the S-9 fraction, the microsomal pellet, and postmicrosomal supernatant from rat livers. The isotope effect (ratio of maximal rates for the deuterated and light substrates) using the S-9 from Long-Evans rat livers was found to be 1.82 for dimethylnitrosamine and 5.38 for phenylmethylnitrosamine. Phenobarbital was shown to induce dimethylnitrosamine demethylase activity in the microsomal pellet of both Long-Evans and Sprague-Dawley rats but to repress this activity in the postmicrosomal supernatant in the Long-Evans rats, while markedly increasing it in the Sprague-Dawley rats. It was also found that there was nitrosamine demethylase activity in the so-called "pH 5 enzymes" and in the supernatant from that preparation. The latter activity shows substantially different characteristics from that found in the other fractions.


Covalent binding of carbaryl (1-naphthyl-N-methyl-carbamate) to rat liver microsomes in vitro. Enzyme mediated binding of carbaryl (1-naphthyl-N-methylcarbamate) to rat hepatic microsomes occurred in vitro in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen and was inhibited by nitrogen, carbon monoxide, reduced glutathione, cysteine, N-ethyl maleimide, and 2-diethylaminoethyl-2,2'-diphenylvalerate-HCl (SKF 525-A). Binding was markedly increased, 2- to 3-fold, by pretreatment of animals with phenobarbital or 3-methylcholanthrene and appeared to be dependent on the production of an active metabolite which was formed oxidatively in liver microsomes by cytochrome P-450 mixed-function oxidases. SDS-polyacrylamide disc gel electrophoresis of ether-extracted, solublized microsomes and the distribution of radioactivity after Pronase digestion, treatment with 1-fluoro-2,4-dinitrobenzene, and extraction with ethyl acetate and ether at pH 9 and 1 indicated that the radiolabeled products were covalently bound to amino acid residues of microsomal protein.


The early effects of chemical carcinogens on adult rat hepatocytes in primary culture: I. Quantitative changes in intracellular enzyme activities following a single dose of carcinogen. The effects of exposure of adult rat hepatocytes to chemical carcinogens have been studied using a short-term maintenance culture system. Scanning microdensitometry was used to quantitate the observed changes in enzyme activity. The dose-response curves showed a biphasic response for all 4 enzymes studied (glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH oxidase and gamma-glutamyl transpeptidase) there being decreased enzyme activities at the higher dose levels used, possibly indicating cytotoxicity. The enhancement of enzyme activity at low dose levels was due to generalised increases occurring in every cell, rather than to selection of a cell species particularly high in enzyme activity. A culture period of 24 h was necessary for the complete adaptation of the cells to the culture environment as evidenced by the response of intracellular glucose-6-phosphate dehydrogenase activity to carcinogen treatment. These findings are discussed in relation to previously reported in vivo studies.


Reactions of nitrosobenzene with reduced glutathione. Nitrosobenzene (NOB) formed acid labile conjugates with reduced glutathione (GSH) and hemoglobin within red cells. In vitro, NOB rapidly reacted with GSH with formation of phenylhydroxylamine (PH), oxidized glutathione (GSSG), and a water-soluble compound identified as glutathionesulfinanilide (GSO-AN). Free aniline (AN), aminophenols and azoxybenzene were not detected. The proportion of PH formed increased with increasing GSH concentration and at higher pH values. Spectroscopic analysis revealed the formation of a labile adduct following a second order reaction (K = 5 x 10(3) M-1 . sec-1 at pH 7.4 and 37 degrees). This reaction was reversible because nearly all NOB could be extracted with ether from the labile intermediate. On the other hand, the labile intermediate was transformed into GSO-AN (with increasing rate at lower pH values) or it was cleaved by GSH with formation of GSSG and PH. Intermediate formation of NOB and thiol radicals was ruled out by analysis of the equilibrium data. A tentative scheme is presented for the proposed reaction mechanism.


The effects of dopamine, bromocriptine, lergotrile and metoclopramide on prolactin release from continuously perfused columns of isolated rat pituitary cells. The control of secretion of prolactin was studied using continuous perfusion of a column of isolated rat pituitary cells supported by Bio-Gel polyacrylamide beads. Prolactin secretion was inhibited repeatedly by dopamine and rapidly recovered in its absence. Maximum inhibition was achieved at 5 x 10(-7) M dopamine. Bromocriptine and lergotrile directly inhibited prolactin release from the pituitary cells. Bromocriptine had a longterm action in inhibiting secretion. The dopamine receptor blocking agent, metoclopramide, overcame the inhibitory effect of dopamine but had no effect on prolactin secretion in its absence.


Adult metachromatic leukodystrophy. Arylsulphatase-A values in four generations of one family and some reflections about the genetics. The authors describe an investigation of Adult Metachromatic Leukodystrophy in a Dutch family, of which two persons were affected. The studies of leukocyte arylsulphatase-A activity were made in 47 members of 4 generations of the same family. The propositus, a 30-year old man, showed a conspicious organic brain syndrome, that progressed in two years to a complete dementia. His leukocyte, liver and kidney arylsulphatase-A activities (ASA) were very low; leukocyte-ASA activity increased after aceto-salicylate. His brother had died at 34 years, after a progressive debelitating neuropsychiatric illness of eight years; postmortem metachromatic leukodystrophy was diagnosed. In all living family members, urine and leukocyte arylsulphatase-A activities were determined. The findings are discussed in relation to the genetics and pathogenesis of this adult form of metachromatic leukodystrophy. Allelic heterozygoty is proposed as inheritance model in this family. Suggestions for further research are made.


Methodology for demonstrating sustained efficacy of hypnotics: a comparative study of triazolam and flurazepam. To test for sustained hypnotic efficacy, triazolam (0.6 mg) or flurazepam (30 mg) was given to chronic insomniac patients for 7 consecutive nights in parallel, double-blind design. Triazolam at this dose was an effective hypnotic by all usual subjective measures and did not produce appreciable hangover. Flurazepam performed similarly. For either drug, comparison of the mean scores for the first 2 nights with that for the last 2 nights for any of the parameters did not reveal any significant difference. Thus, both triazolam and flurazepam showed sustained efficacy for 1 week at these doses. Some interesting theoretical and practical questions about the measurement of sustained efficacy of hypnotics in situations of repetitive dosing were addressed by the study. While a placebo control is desirable, the results obtained may be uninterpretable. An acute-care hospital setting may not be the ideal setting for doing such studies. There were indications from the study that the first-night results in a hypnotic clinical trial may be atypical.


Changes in the state of ionization of carboxyl groups in elastin in response to the binding of sodium dodecyl sulfate. The interaction of sodium dodecyl sulfate with elastin has been studied by complexometric titration. Approximately 1.2 mumoles of protons with a pKapp of 5.45 are taken up by 10 milligrams of insoluble elastin upon the binding of detergent, apparently due to the protonation of normally ionized carboxylate functions in this protein. Since ionized carboxylate functions of elastin are essential for its interaction with elastase and, possibly, metallic cations, these results may have physiological significance in view of the affinity of elastin for lipid-like ligands.


Cutaneous-systemic reactions to toxins and venoms of common marine organisms. The potential adverse effects of North American marine organisms are presented herein. It is stressed that water sports enthusiasts should be aware of possible dangers when in unfamiliar waters.


Nonthrombocytopenic purpura induced by carbromal. A case of carbromal-induced purpura is described and the typical clinical features are presented. The diagnosis was confirmed by patch tests with 1 percent and 5 percent carbromal in propylene glycol and the positive results of a macrophage migration inhibitory factor test for carbromal. The rash gradually subsided following withdrawal of the drug.


Effects of ethanol on plasma protein shedding in the human stomach. Plasma protein shedding in the stomach was measured in 23 normal individuals before and after intragastric administration of a 30% solution of ethyl alcohol. Two different methods were used to assess plasma protein shedding. The first technique utilizes [131I]albumin and requires neutralization of the gastric juice. It was used in 12 subjects and failed to demonstrate any increase of plasma protein shedding under the influence of ethanol. The second technique which utilizes [51Cr]chloride was used in 11 subjects. It demonstrated a significant increase of the gastric clearance of plasma protein which reached 2.5 times the control values. The [51Cr]chloride technique does not require prior neutralization of gastric acidity. It is concluded that, in normal man, ethanol administration increases plasma protein shedding in the stomach when it is given in the presence of an acid gastric juice. The effect is not observed when the gastric acidity is neutralized.


Continuous peritoneal dialysis as treatment of acute experimental pancreatitis in the rat. II. Analysis of its beneficial effect. In acute sodium-taurocholate-induced pancreatitis in the rat, peritoneal dialysis reduced serum amylase levels and the amount of fat necrosis, but did not influence the damage to the pancreas itself. Pancreatic ascites obtained in the early course of the disease was found to have a hypotensive effect when given intraperitoneally to healthy rats. This effect vanished in the later course of acute experimental pancreatitis and was reduced by acidification of the ascites or by administration of an antihistaminic drug. Thus the beneficial effect of continuous peritoneal dialysis on survival time and mortality rate seems to be of systemic origin.


[Comparison of the metabolism of isolated streptococci and dental plaque]. 1. Isolated cultures of Streptococcus mutans form lactic acid as well as small amounts of formic acid and acetic acid during the anaerobic break down of glucose. The composition of the acids in terms of percent is independent of time. 2. Plaque specimens incubated in vitro show a complicated spectrum of substances; propionic and butyric acids are found in addition to lactic, formic, and acetic acids. The composition in terms of percent depends on the incubation period. The percentage of lactic acid drops as the incubation period is extended. 3. Just as the plaque, the product spectrum of isolated streptococci in terms of quantitative composition depends on the buffer capacity.


Thyrotropin binding to cultured lymphocytes and thyroid cells. The TSH-binding properties of human lymphocytes in continuous culture were studied and compared to those of bovine and human thyroid cells in primary culture. Both lymphocytes and thyroid cells had maximal TSH-binding capacity at pH 5.2. At pH 7.4, thyroid cells bound 15% but lymphocytes bound only 3% of the amount bound at pH 5.2. At 37 C, maximal binding of [125I]iodo-TSH to lymphocytes was reached within 60--90 min and maximal binding to thyroid cells was reached within 15--20 min. TSH binding to lymphocytes was salt sensitive, being inhibited to 50% by 0.2 mM MgCl and 0.4 mM CaCl2 and by 20 mM Kl, KCl, and NaCl. The saturable binding of bovine TSH (bTSH) to thyroid cells at pHs 5.2 and 7.4 was above 90% of the total binding. Saturable binding of bovine TSH (bTSH) to thyroid cells at pHs 5.2 and 7.4 was above 90% of the total binding. Saturable binding to lymphocytes at pH 5.2 was also above 90%, but at pH 7.4 was 75% of total. At pH 5.2, both cell types displayed identical displacement curves of [125I]iodo-bTSH by unlabeled bTSH. Pure hCG, human placental lactogen, human GH, and insulin cross-reacted to less than 1% with [125I]iodo-bTSH binding to lymphocytes at pH 5.2, whereas a crude preparation of hCG and human FSH plus human LH showed a strong cross-reaction. Nonhormone glycoproteins, including mucin, normal human gamma-globulin, and bovine thyroglobulin showed intermediate cross-reactivity. At pH 7.4, the cross-reactivity of normal human gamma-globulin, bovine thyroglobulin, and pure hCG with bTSH binding to both lymphocytes and thyroid cells was below 1%. The TSH-binding properties of lymphocytes and thyroid cells show many similarities but differ in kinetics and the relative binding capacity at neutral pH. Although the physiological significance of these differences is not yet clear, cultured cells provide a convenient system for studies of TSH-receptor interaction.


Testicular teratoma in an equine cryptorchid. An abnormal cryptorchid testicle removed from the abdominal cavity of a 4 year old Thoroughbred stallion is described. The abnormal organ conforms to the requirements of Willis (1960) for a teratoma. The difference between these tumours in man and horses is discussed.


Blood-gas, acid-base and haematological values in horses during an endurance ride. The effects of prolonged strenuous exercise on arterial and venous oxygen tension, carbon dioxide tension, pH, bicarbonate, standard bicarbonate, base excess, haemoglobin, packed cell volume and total plasma protein were studied in 36 horses during a 100 km endurance ride. There were significant changes in many parameters when pre-ride values were compared with both mid-ride and end of ride values. The prominent changes were the development of dehydration and a metabolic alkalosis. At the mid-ride sampling time those horses with higher heart rates had a greater degree of metabolic alkalosis than those with lower heart rates. The first 4 horses in the race completed the ride with speeds between 322-330 m/min and demonstrated a metabolic acidosis.


Response of erythrocytic 2,3-diphosphoglycerate to strenuous exercise. Since increases of erythrocytic 2,3-diphosphoglycerate (2,3-DPG) have been shown to enhance the release of oxygen from hemoglobin, experiments were designed to evaluate the response of 2,3-DPG to two different work-loads in 13 fasted human subjects. No signficant mean change in 2.3-DPG was found following 16 min of strenuous exercise on a bicycle ergometer, but when the subjects were subjected later to a greater workload for 20 min, there was a significant mean decrease in 2.3-DPG despite much individual variation. In addition, there was a significant positive correlation of 2.3-DPG reduction with increases in postexercise lactate, and a significant inverse correlation of oxygen consumption during exercise with postexercise lactate. The data suggest that the 2.3-DPG mechanism may not be compensating in exercise when the workload requires a preponderance of anaerobic metabolism promoting lactacidaemia.


Effects of active, passive or no warm-up on the physiological response to heavy exercise. Six endurance-trained young men were subjected to a 4 min maximal aerobic treadmill run (100% of VO2 max), after active or passive warm-up or rest on separate days. The increase in body temperature during the active and passive warm-up was controlled, so that the temperature reached the same level, before the subject was exposed to the maximal exercise. On average the rectal temperature rose to 38.3 degrees C (range 38.1-38.6 degrees C). The standard work resulted in a significant higher oxygen uptake, lower lactate concentration and higher blood pH when the work was preceded by active warm-up as compared with passive or no warm-up. The difference in total oxygen uptake during the run between the active and passive warm-up procedure was 0.8 1. No significant difference in minute volume of expired air or respiratory quotient was found. It is concluded that the physiological effects of a thorough active warm-up may be of substantial benefit to athletic performance.


A 13C nuclear-magnetic-resonance study of CO2-HCO3-exchange catalyzed by human carbonic anhydrase C at chemical equilibrium. The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of 13CO2 and NaH13CO3 were measured at 67.89 MHz. Enzyme-catalyzed CO2-HCO-3 exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with 1H2O or with 2H2O as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady-state Km values are assumed to represent substrate dissociation constants. The main conclusion concerning the catalytic mechanism of the enzyme is that the proton-transfer processes which limit turnover rates in the steady state are not directly involved in CO2-HCO-3 exchange. In addition, the results suggest that CO2-HCO-3 interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH- with CO2.


Conformational studies of neurotoxin II from Naja naja oxiana. Selective N-acylation, circular dichroism and nuclear-magnetic-resonance study of acylation products. After treatment of neurotoxin II, a component part of the venom of the Middle Asian cobra Naja naja oxiana, with acetoxysuccinimide all five possible epsilon-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacetyl)-neurotoxins II and the hexa(trifluoroacetyl)-neurotoxin II, which were studied by circular dichroism (CD), 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five epsilon-amino groups) in the hexa(trifluoroacetyl)-neurotoxin II NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the 19F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin II 19F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.


Synthesis and activation of asparagine in asparagine auxotrophs of Saccharomyces cerevisiae. L-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms. Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA- and asnB-. Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutationalone. This, and Jones' report [J. Bacteriol. 134, 200-207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in sigma 1278b wild type) is cancelled by casnI- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant.


Spin transition of camphor-bound cytochrome P-450. 2. Kinetics following rapid changes of the local paH at sub-zero temperatures. The kinetics of the spin transition in the heme iron of the cytochrome P-450 substrate complex have been observed by stopped-flow measurements between 4 degrees C and -27 degrees C. Large displacements in the spin equilibrium are induced by small changes in the concentration of hydrogen or potassium ions. The kinetic and thermodynamic data indicate that the spin transition is rate-limited by conformational changes of the protein. The spin transition appears to be governed by the local paH, modulated in turn by external factors: a satisfying kinetic analysis is attained only by accounting for the difference between local paH (paH, in) and bulk paH (paH, out), as described in the preceding paper. Indeed, the low-spin to high-spin rate constant obeys a local paH titration curve with a pK = 5.4 +/- 0.1 at -17 degrees C.


Solution conformation of glycosaminoglycans: assignment of the 300-MHz 1H-magnetic resonance spectra of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate, and investigation of an alkali-induced conformation change. Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl beta-D-glucopyranosiduronate, methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution. As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter-residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H-2 of the glucuronate residue, and the CH3 and H-2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts. Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.


Investigations on the activation of bovine prochymosin. Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.


Electrophysiologic properties of intravenous Tenormine in man. The acute electrophysiologic effects of intravenous Tenormine (0.1 mg/kg body weight), a new cardioselective beta-adrenoreceptor blocking drug, were studied in 18 subjects with estimated normal impulse formation and conduction. The most significant (P less than 0.01) effects were sinus cycle lengthening, depression of intranodal conduction and prolongation of AV node refractory periods. Sinus node recovery time, sinoatrial conduction time and atrial refractory periods were only slightly prolonged (P less than 0.05). Intraatrial conduction and infra-His conduction were unchanged. These properties are compared with those of the most commonly employed beta-blocking agents. The clinical implications are discussed.


[The measurement of the placenta, umbilical cord and components of amniotic fluid in beagle dogs]. The body weight of newborns, the weight of placenta, the length of umbilical cord and the content of urobilinogen, gulcose, protein, pH, keton body in amniotic fluid were investigated with samples from 16 litters of beagles. The mean body weight of newborn, weight of placenta and length of umbilical cord were 258.0 +/- 44.6 g (85 samples), 48.5 +/- 8.5 g (85 samples) and 11.0 +/- 2.7 cm (52 samples), respectively without any sex difference. The significant correlation were recognized between the weight of placenta and the body weight of newborn, between the length of umbilical cord and the body weight of newborn and between the weight of placenta and the length of umbical cord (P less than 0.01).


In vitro proliferation of haemopoietic cells in the presence of adherent cell layers. I. Culture conditions and strain dependence. The culture system, in which a marrow-derived adherent cell population, established in vitro, exerts a long-term promoting influence on proliferation of haemopoietic cells, is reproduced. Essential parameters of the system are investigated; it is confirmed that the system is critically dependent on horse serum, and on the in vitro age of the adherent cell layer. The growth-promoting effect on haemopoietic cells seems to be independent of the number of marrow cells per culture flask initially inoculated into the cultures to establish the adherent cell layer. In vitro established marrow-derived adherent cell layers from RFM (H-2f) and BALB/c (H-2d) mice can promote the long-term proliferation of syngeneic and allogenic haemopoietic cells; haemopoietic marrow cells from C3H (H-2k) cannot be maintained on syngeneic or allogeneic (BALB/c, H-2d) adherent cell layers; adherent cell layers of C3H (H-2k) can maintain haemopoietic cells of the H-2d (BALB/c) genotype. This culture system does not reproduce the in vivo phenomenon of allogeneic resistance. The relevance of these findings to the suggestion that the growth-promoting activity of adherent marrow cells on haemopoietic stem cells in vitro duplicates aspects of the in vivo haemopoietic microenvironment is discussed.


Induction of spermatogenesis in the naturally cryptorchid pig. Abdominal testes of adult, naturally unilaterally cryptorchid boars were subjected to continuous artificial cooling for 5, 15, 25, or 45 days. This treatment initiated development of the spermatogenic epithelium. After a cooling period of 45 days there was complete differentiation in many seminiferous tubules. The results indicate that the spermatogenic arrest in abdominally located testes is not due to an inborn defect in the testis, but is caused by the maintenance of the testis at the abdominal temperature.


Axoplasmic transport of noradrenaline in the sciatic nerves of spontaneously diabetic mice. The intraaxonal transport of noradrenaline and tyrosine hydroxylase was studied in the sciatic nerves of diabetic, obese and appropriate control mice. Noradrenaline and tyrosine hydroxylase accumulated proximal to a constriction applied to the nerves. Noradrenaline concentration in the non-constricted sciatic nerves did not differ significantly in nondiabetic and diabetic mice (0.67 +/- 0.04 and 0.58 +/- 0.01 ng/cm respectively); in obese mice and lean littermates the noradrenaline concentration was 0.47 +/- 0.05 ng/cm and 0.46 +/- 0.01 ng/cm. After nine hours of constriction the concentration of noradrenaline increased in the axons of nondiabetic (1.15 +/- 0.06 ng/cm), in obese (0.90 +/- 0.08 ng/cm) and lean mice (1.10 +/- 0.07 ng/cm) but remained low (0.68 +/- 0.07 ng/cm) in diabetic mice.--Administration of insulin (10 U/kg/day) to diabetic mutants completely reversed the decrease in NA accumulation. NA accumulated only in the nerve segment proximal (1 cm) to a constriction and was transported distally at an apparent velocity of 0.75 mm/hr in control axons. The difference of NA accumulation between diabetic and nondiabetic control indicate a reduced rate of axoplasmic flow in the noradrenergic axons of diabetic animals.


Insulin receptors in cultured mouse retinal cells. The binding of 125I-insulin to uncloned and cloned cultures of mouse retinal cells has been investigated. At 15 degrees C, binding of the hormone reached a steady state by 60 min, while at 37 degrees C equilibrium was reached earlier but at a lower level than at 15 degrees C. Porcine insulin, porcine proinsulin and guinea pig insulin displaced labelled insulin in proportion to their known biological potency. A sharp pH dependence of the hormone binding was observed with an optimum at pH 7.8. The dissociation rate of the 125I-insulin was increased in the presence of unlabelled hormone, suggesting the existence of negative cooperatively in the insulin-receptor interaction. The availability of established retinal cell lines with insulin receptors should facilitate the study of the insulin-retina interactions in a controlled in vitro system.


Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine. A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on  monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.


Release of gastric inhibitory polypeptide (GIP) by intraduodenal acidification in rats and humans and abolishment of the incretin effect of acid by GIP-antiserum in rats. Intraduodenal infusion of 0.05-0.5 N hydrochloric acid dose-dependently increases serum levels of immunoreactive gastric inhibitory polypeptide (GIP) in rats. Immunoreactive GIP released by duodenal acidification is biologically active because it augments the glucose-induced release of immunoreactive insulin (IRI). This augmentation of glucose-induced IRI release by intraduodenal acid can be abolished for 30 min by simultaneous intravenous infusion of GIP-antiserum. From this it is concluded that the initial capacity to augment the glucose-induced insulin release (incretin activity) of hydrochloric acid is due to its ability to release GIP. Later on, other gut factors with incretin activity might be released by hydrochloric acid. Also, in humans, intraduodenal infusion of 0.1 N hydrochloric acid releases GIP without changing serum levels of glucose or insulin. The GIP release is a direct effect of intraduodenal acid and is not mediated via secretin release. Injection of secretin in supraphysiologic doses does not change serum levels of immunoreactive GIP. However, such secretin injections induce a short-term insulin release and a decrease in serum glucose concentration.


Hepatocellular injury with distinctive mitochondrial changes induced by lergotrile mesylate: a dopaminergic ergot derivative. Increased serum activities of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) occurred in 12 out of 19 patients with idiopathic parkinsonism when they were treated with the ergot derivative lergotrile at an oral dose varying from 50 to 150 mg daily. Hepatocellular injury was confirmed by microscopic examination of liver biopsies obtained from 3 of these patients when the serum activities of ALT and AST were appreciably elevated. Light microscopy revealed features of mild acute hepatocellular injury, and electron microscopy showed proliferation of the smooth endoplasmic reticulum and apparently unique mitochondrial changes in hepatocytes. This is the first report of pathological changes in the liver associated with the therapeutic use of an ergot derivative. The presence of a potentially reactive cyanide group in the lergotrile molecule could be causally related to the observed hepatocellular injury. It is suggested that serum ALT and AST activities should be monitored carefully when the therapeutic potential of any new ergot derivative is assessed.


Evidence for different forms of human chorionic gonadotropin receptor complex in pseudopregnant rat ovaries. After in vivo application of [125 I]-iodo-hCG to pseudopregnant rats, radioactivity extracted from ovarian particulate fractions can be eluted from Sephadex G--200 columns in three various fractions (Vo, Kav = 0.15, Kav = 0.27) differing from hCG (Kav = 0.35). By lowering the pH 50% of the radioactivity is precipitated and the resuspended material shows a conversion to the smaller complexes. The supernatant material is dissociated to [125I]-iodo-hCG. In alkaline milieu [125I]-iodo-HCG is dissociated completely from the complexes.


A light and electron microscopic study of an ovarian and rectal carcinoid. An ovarian and rectal carcinoid within the same person is described. The ovarian carcinoid is non-argentaffinic with typical regular granules. The rectal tumour consists of two types of APUD cells, some argentaffinic and diazo-positive, whilst others are non-argyrophilic. These features are mirrored ultrastructurally, some cells containing the large pleomorphic argentaffinic granules while others have smaller regular granules. In addition, focal aggregates of microfilaments were found in both tumour types. These findings are discussed in terms of the APUD concept. The similarities of the APUD tumour cells to D1 APUD cells of the duodenum is highlighted, and an alternative explanation for their derivation is proposed.


Exposure therapy for phobias and obsessive-compulsive disorders. When abnormal fears or excessively compulsive behavior begin to rule a person's life, the consequences can be devastating to both patient and family. But three quarters of cooperative patients can be restored to normal functioning through a form of therapy based on exposing the patient continually to the stimuli that evoke symptoms until they can be tolerated and the phobic or compulsive behavior dissipates.


Oxidation of the sulfhydryl forms of insulin A-chain and B-chain. A modified procedure for the preparation of the S-sulfonates of the A- and B-chains of insulin and their conversion to the sulfhydryl forms by tri-n-butylphosphine is described. Air oxidation of the sulfhydryl forms of the A-chain in dilute solution (0.2 mg/ml) either in the presence or absence of urea at pH 9.0 yields primarily monomeric, intrachain disulfides. Similar treatment of the reduced B-chain yield monomeric, intrachain disulfide in 7 M urea but a large number of oligomeric, interchain disulfides in the absence of urea. Electrolytic reduction of insulin in 7 M urea of pH 8.5, followed by oxidation of the sulfhydryls in dilute solution in 7 M urea at pH 9.0 yields primarily a mixture of the monomeric, intrachain disulfides of the A-chain and of the B-chain which can be separated by chromatography on Sp-Sephadex in acidic urea. The rate of the oxidation of the sulfhydryls of the two separate chains was much slower and less complete than that reported for the two chains crosslinked by the carbonylbismethionyl residue.


Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis. LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.


Maleylation and pH-dependence of Streptomyces griseus protease B. The enzymatic activity of Streptomyces griseus protease B (SGPB) was measured over pH range 8.4--11.5 using a specific new, chromophoric substrate N-succinyl-glycyl-glycyl-L-phenylalanine p-nitroanilide. It was found that the activity is dependent on ionization of a single group with apparent pK = 10.84, possibly lysine-125. Maleylation of the epsilon-amino group of this lysine was linearily associated with the loss of enzymatic activity. It is therefore suggested that the electrostatic interaction between the side chain of lysine-125 and the alpha-carboxyl group of the C-terminal tyrosine is crucial to the active conformation of the enzyme. In contrast the maleylation of the alpha-amino group of the N-terminal isoleucine was rapid but could not be correlated to the loss of activity.


pH dependence of the circular dichroic bands of phenylmethanesulfonyl-mesentericopeptidase. The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl-mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2--10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH-dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the "exposed" tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.


Kinetic studies on human cysteinyl-tRNA synthetase. The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis.


The pH tolerance of rabbit and human corneal endothelium. The endotheliums of rabbit corneas were perfused in an in vitro perfusion specular microscope up to 3 hr with solutions varying in pH from 3.5 to 10.0. Corneal thickness was monitored throughout the experiment, and at appropriate times the corneas were prepared for SEM and TEM. Analysis of the corneal thickness data and interpretation of the electron micrographs reveals that outside of the pH range of 6.5 to 8.5, structural and functional alterations occur. Direct cellular damage, as well as disruption of junctional complexes, lead to a breakdown in the barrier function of the corneal endothelium. The extent of this breakdown is dependent upon both the magnitude of the pH change and the exposure time. Further experiments on banked human eyes support this finding.


Attachment of bacteria to exfoliated cells from the urogenital tract. To establish urogenital infections, organisms must adhere to the mucosal lining. A differential adherence capacity among various bacterial species was observed when exfoliated urethral and urothelial cells were tested in an in vitro system. No difference in the adherence capacity of a particular species was observed when tested with exfoliated cells obtained from voided urine from different healthy individuals of the same sex. Escherichia coli harvested directly from urine specimens of patients with significant bacteriuria showed a significantly higher capacity to adhere than when obtained from the primary isolation plate. Staphylococcus saprophyticus adhered significantly better to urothelial cells than did Staphylococcus epidermidis. Adherence did not differ when the tests were performed in ultrafiltrated, infected and noninfected urine. Variations of the osmolality did not influence the adherence rate of E. coli. Gonococci showed an increased capacity to adhere when tested in urine of increasing acidity. Gonococci producing T1 colonies adhered by significantly higher numbers per cell than such bacteria producing T4 colonies.


Rapid screening for urease inhibitors.

Rapid screening for urease inhibitors. Two methods are described that are suitable for the rapid screening of compounds as urease inhibitors. The first utilizes an electrode sensitive to NH4+ ions; the second is dependent on pH rise. A feature of both is a direct readout of reaction rate.


Observations on the gubernaculum during descent of the testis. The mechanisms that influence the descent of the testis are not clearly understood. The gubernaculum is a structure worthy of scrutiny inasmuch as it is conspicuous during descent, but virtually disappears after descent is complete. Early in gestation, the rat gubernacular bulb consists of loose mesenchymal cells that develop into fibrillar cells. These later thicken into rhabdomyoblasts that, near the end of gestation, differentiate into spiral striated muscle bundles, and eventually migrate outward into the abdominal/scrotal wall. The rhabdomyoblasts of the female gubernaculum do not differentiate further but rather undergo fatty degeneration. It is possible that spiral contractions of the attached gubernaculum produce tension on the testis and induce descent. The gubernaculum as the receptor organ for testicular descent may be responsive to local testicular hormones. Likely candidates are testosterone, dihydrotestosterone, or Mullerian Inhibiting Substance. A thorough knowledge of the sequential differentiation of the gubernaculum during embryonic development sets the stage for the study of its response to hormonal manipulation both in vivo and in vitro.


Some properties of RNA polymerases of different influenza viruses. The transcriptases of several influenza viruses were tested for pH and temperature dependence and for thermal stability. Marked differences in pH dependence and thermostability were determined.


Non-routine extractions in orthodontic treatment. A four-unit symmetrical premolar extraction case demands meticulous levelling, overjet reduction, space closure, rotating, paralleling and torqueing to justify the gambit of having extracted the teeth at the commencement of treatment. This involves the patient in complex therapy which may, for any of several reasons, be contra-indicated for that particular patient, though a decision based on the plaster casts alone may have upheld such an approach. The purpose of this article is to discourage automatic decisions to extract first premolars in orthodontic extraction cases. The clinician is offered a classification of special cases in which an alternative should be sought, based on careful consideration of the general dental, facial, physical, psychological and economic state of the patient.


The pH dependence of borohydride as an aldehyde reductant. The aldehyde-reducing capacity of borohydride has been investigated in the sequence periodic acid-borohydride-periodic acid-Schiff and variants. Densitometric studies on rat colonic mucins show that borohydride incompletely blocks periodate-engendered aldehydes unless the pH is above 8.2. Below this value, some aldehydes are not reduced and continue to be Schiff-stainable, while others are subsequently gerenated by the second exposure to periodic acid. The effect is more pronounced in paraffin than in cryostat sections, but does not apply to human colonic mucins.


A quantitative cytochemical method for the demonstration of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary. A method for the quantitative measurement of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD+ or NADP+ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP+ dependence is not abolished by inhibiting nonspecific alkaline phomonoesterase. The activity of delta5,3beta-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10(-4) M.


Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH. The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres. In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.


Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides. A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.


Differences of affinity and energetics of the active transport systems for amino acids in Chang liver cells. The plasma membrane of Chang liver cells was shown to have at least two distinct active transport systems, one with preferential affinity for glycine and one for leucine. The uptakes of glycine and leucine were specificially inhibited by Me-AIB and b-BCH, respectively. The uptake of glycine decreased remarkably within 10 min on incubation with DNP (2 mM), KCN (5 mM), and malonate (20 mM) under aerobic conditions, along with a decrease of cellular ATP concentration to as low as 1/4 of normal, while the uptake of leucine was not depressed under these conditions. Leucine uptake was, however, greatly reduced within 10 min on incubation with DNP plus ICH2CONH2 (5 mM), when the cellular ATP was estimated at about 0.066 mM. The active transport of leucine, but not that of glycine, was accompanied by further acidification of the intracellular fluid, which was lower in pH than the extracellular fluid by approximately 0.3 unit without addition of amino acid to the medium.


Pterin deaminase from Bacillus megaterium. Purification and properties. A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial extract. The specific activity was raised 90-fold over that of the crude extract. The pH optimum is around 7.3, and the Km value for 6-carboxypterin is 1.3 mM. The molecular weight of the enzyme was estimated by gel filtration to be about 110,000. The enzyme deaminated pterin, 6-carboxypterin, biopterin, 6-methylpterin, 7-methylpterin, xanthopterin, 6-hydroxymethylpterin, sepiapterin, isosepiapterin, folic acid, and 6,7-dimethylpterin to their corresponding lumazines, whereas guanine, 7-carboxypterin, leucopterin, isoxanthopterin, and 6-methylisoxanthopterin did not serve as substrates. The enzyme was inhibited by PCMB and 8-azaguanine.


Studies on the delta 5-desaturation in ergosterol biosynthesis in yeast. Studies were carried out on the delta 5-desaturation reaction in ergosterol biosynthesis with a particulate fraction of cell-free extract of yeast. A reduced pyridine nucleotide coenzyme and molecular oxygen were required for the reaction. It was shown that the enzyme activity is located in a fraction corresponding to microsomes. The reaction was inhibited by KCN, but not by CO. Menadione and potassium ferricyanide inhibited the NADPH- and NADH-dependent reactions, respectively, and cytochrome c inhibited both of them. These results suggested an involvement in delta 5-desaturation of a mixed function oxidase system resembling that for the fatty acyl-CoA desaturation reaction.


Inhibitory effects of nucleoside triphosphates on nucleolar RNA synthesis. Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.


Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate. It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).


The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation. The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.


Interaction of lactoperoxidase with thiols and diiodotyrosine. Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.


Some properties of Escherichia coli glutamine synthetase after limited proteolysis by subtilisin. Escherichia coli glutamine synthetase is inactivated by subtilisin. Protection against inactivation is afforded by glutamine and ammonium ions. One large fragment (Mr = 35,000) is identified by sodium dodecyl sulfate-gel electrophoresis and carries adenylylation site. Smaller quantities of two other fragments (Mr = 17,000 and 15,000, respectively) are als observed oo observed on the gel. tthe nicked protein remains dodecameric, as evidenced by electrophoresis and centrifugation. It has retained the binding properties toward ADP and Ci-bacron blue and undergoes conformation changes upon binding, as does the intact protein. It is recognized by the antiserum raised against the native enzyme. The nicked protein also remains an excellent substrate of E. coli adenylyltransferase.


Limited proteolysis of glutamine synthetase is inhibited by glutamate and by feedback inhibitors. Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.


Oxidation of horseradish peroxidase compound II to compound I. In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.


The mechanism of the skeletal muscle myosin ATPase. III. Relationship of the H+ release and the protein absorbance change induced by ATP to the initial Pi burst. Several phenomena are associated with the binding of ATP to myosin: 1) a fluorescence enhancement, 2) a release of H+, and 3) a protein absorbance change. In the accompanying paper (Chock, S. P., Chock, P. B., and Eisenberg, E. (1979) J. Biol. Chem. 254, 3236-3243), it was demonstrated that the fluorescence enhancement is mainly caused by the hydrolysis of ATP in the initial Pi burst rather than by the conformational change induced by the irreversible binding of ATP. In the present study, the cause of the H+ release and the protein absorbance change were investigated. The results show that like the rate of the fluorescence enhancement the rates of the H+ release and the protein absorbance change level off at high ATP concentration at a much lower rate than the rate of irreversible ATP binding. Furthermore, under all conditions tested, the rates of the H+ release and the protein absorbance change are equal to the rate of the initial Pi burst. Therefore, like the fluorescence enhancement, most of the H+ release and the protein absorbance change are associated with the initial Pi burst rather than the binding of ATP.