Electromyograph biofeedback as an adjunct to standard psychiatric treatment. The effectiveness of electromyograph (EMG) biofeedback training was studied among 2 groups of psychiatric patients, and a control group of normal subjects. Both the psychiatric patients and the normal subjects received 6 sessions of training in deep muscle relaxation and biofeedback. The schizophrenic and psychoneurotic patients had significantly lower ratings for pathological symptomatology and abnormal behavior and all groups significantly reduced their level of muscle tension following the treatment; the schizophrenics and normals displayed significantly greater reductions than the psychoneurotic patients. The length of hospitalization for the 2 groups of patients in the study was lower than for similar patients who received standard psychiatric treatment but no biofeedback training. These results suggest that EMG training can be a beneficial adjunctive treatment for severely disturbed patients.


An Isakower phenomenon variant in an initital dream. The Isakower phenomenon is reviewed, and a psychotherapy case is described. The first 2 dreams of the therapy are felt to be variants of the Isakower phenomenon, and are believed to support several hypotheses in the literature concerning the phenomenon.


Brief reactive psychosis: a psychodynamic interpretation. The diagnosis "brief reactive psychosis" leaves little implied in the way of etiology or treatment approach. Three case histories are presented to illustrate the term, followed by brief summaries of the major dynamics involved. The older diagnostic term "hysterical psychosis" is discussed, and a parallel is drawn between this form of brief reactive psychosis and others so that they may be psychodynamically understood and that treatment may be rationally based upon this model.


Case studies in neuropsychiaatry. II: Conversion pseudodementia. A patient is described who experienced pseudodementia as a conversion reaction. The patient was considered to be demented originally because of the evidence for profound cognitive impairment elicited on mental state examination. Careful analysis of the patient's verbal production  in unstructured situations and of her behavior inthe hospital permitted a clinical diagnosis of pseudodementia to be made, a diagnosis confirmed by response to treatment and long term follow-up. Evidence is presented to support the interpretation of her pseudodementia as a conversion reaction.


Renal damage associated with long term use of lithium carbonate. The authors report 2 cases of renal damage associated with lithium carbonate treatment. The relevant literature is reviewed and supports the claim that the lithium ion may be nephrotoxic. The article concludes with practical recommendations for clinical practice until futher evidence regarding this association is available.


Patterns of drug use in psychiatric inpatient wards. Drug use was monitored in a total of 2,592 psychiatric inpatients in 6 hospitals. The efficacy was rated as satisfactory in 78% of the drug exposures. Nearly 37% of the patients had an adverse reaction. However, only 1.5% of the reactions were noted as being of major severity.


The early manifestations of adolescent thought disorder. Clinicians who wait for disturbed adolescents to fulfil the diagnostic criteria for schizophrenia or a toxic psychosis will delay diagnosing a developing thought disorder until the youngster has been taught to be crazy and has become chronically stunted psychologically. Earlier diagnosis can allow more prompt prevention and support and can facilitate treatment and minimize handicapping. No single symptom is diagnostic of a developing thought disorder but thought disruption can be suspected when a cluster of persistent symptoms is present. These early manifestations of teenage thought disorder are presented and discussed.


The significance of the serum levels of vitamin B-1 and magnesium in delirium tremens and alcoholism. The relation between serum levels of vitamin B-1 and magnesium at admission, and the clinical state was studied in hospitalized patients with either alcoholism or delirium tremens. Vitamin B-1 was deficient in the delirium group, magnesium in both groups. There was no correlation between vitamin B-1 and magnesium levels. Patients with an unfavorable outcome at discharge had lower vitamine B-1 levels and a higher age than patients with a favorable outcome in either group. The magnesium deficiency is discussed for longstanding malnutrition in both groups, the vitamin B-1 deficiency for an unusual high intake of alcohol before delirium tremens started.


Anorexia nervosa and mental retardation: a case report. This is the first published report of a retarded individual developing anorexia nervosa. The authors discuss the interaction between subnormal intelligence and the presentation of anorexic symptomatology. They also discuss the relationship between this disorder and depression. Anorexia nervosa may not be uncommon in retarded adolescents but may go undiagnosed because of the belief that mentally retarded individuals do not develop this disorder.


Investigations on myelination in vitro. Regulation by thyroid hormone in cultures of dissociated brain cells from embryonic mice. Cultures of dissociated brain cells from embryonic mice were used to study the influence of thyroid hormone on myelination in vitro. Synthesis of myelin-associated lipids such as cerebrosides and sulfatides was used as an index for myelination. An experimental design, in which the cells were grown on medium containing serum from a thyroidectomized calf, was employed to demonstrate the direct effect of L-3,5,3'-triiodothyronine (T3Y on the biosynthesis of myelin lipids. The cells grown in the presence of hypothyroid calf serum which contained very low levels of thyroid hormones (T4 (thyroxine), 1.2 microgram/ml; T3, less than 25 ng/100 ml) compared to normal serum (T4, 5.8 microgram/ml; T3, 110 ng/100 ml) showed a diminished synthesis of myelin-associated glycolipids. This reduced activity could be restored to normal by including T3 (13 ng/ml) in the medium.


7-Methylguanosine-dependent inhibition of globin mRNA translation by methylglyoxal. Studies on the inhibition of translation by methylglyoxal of capped and chemically decapped globin mRNAs in the rabbit reciculocyte system strongly suggest that it is cap-dependent. Concentrations of methylglyoxal (0.2 mM), which effected substantial inhibition (80%) of capped mRNA, were only slightly inhibitory (10%) to the decapped species. In addition, the inhibition was K+-dependent with maximal inhibition occurring at the K+ optimum for translation of the capped species, suggesting cap recognition is required for the effect. Results with endogenous mRNA further substantiate that initiation and not elongation is the site of action. These results are consistent with an inhibition due to a newly discovered, rapid reaction of methylglyoxal with the 7-methylguanosine of the cap structure.


Platelet-activating factor. Evidence for 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine as the active component (a new class of lipid chemical mediators). A glyceryl ether containing phosphoglyceride, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (Ac-GEPC), has been shown to have a biological activity indistinguishable from that of naturally generated (rabbit) platelet activating factor (PAF). Its biochemical and biological properties so closely parallel those of naturally occurring PAF that we propose they are one and the same compound. Both PAF and AcGEPC could be converted to an inactive form through base-catalyzed methanolysis and restored to 100% functional activity by reaction with acetic anhydride. The synthetic lipid, AcGEPC, elicited 50% secretion of serotonin from rabbit platelets at a level of 10(-10) M (based on phosphorus). A propionyl derivative had somewhat comparable activity towards platelets, whereas the butyryl homologue was some 7-fold less active and the stearoyl derivative was inactive. These short chain acylglyceryl ether phosphoglycerides represent an entirely new, potent and unique class of lipid chemical mediators. 1-Acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AcLL) also exhibited activity towards platelets but was some 200-fold less active than AcGepc. the propionyl lysolecithin behaved quite similarly to AcLL, but butyryl and stearoyl lysolecithins showed no activity.


Purification of riboflavin-binding proteins from bovine plasma and discovery of a pregnancy-specific riboflavin-binding protein. Riboflavin-binding proteins have been purified from bovine plasma using flavinyl agarose beads. At least three major protein bands, migrating in regions assigned to the beta- and gamma-globulins of plasma, are observed by cellulose acetate electrophoresis. These proteins coelute from a calibrated Sephadex G-100 column in the volume corresponding to a molecular weight of approximately 150,000; a small amount of another riboflavin-binding protein (molecular weight approximately 37,000) is also present. Polyacrylamide gel electrophoresis of the proteins,  with detection by autoradiography of those having tightly bound [2-14C]riboflavin, reveals one protein band which is present only in preparations from pregnant cows. This protein has been purified to apparent homogeneity by storing the mixture of riboflavin-binding proteins at 8 degrees C for 3 weeks, which precipitates the other, less stable proteins. Hence, bovine plasma, like that of the laying hen, contains a number of riboflavin-binding proteins, one of which correlates with pregnancy.


Phosphorylation of ATP citrate lyase in response to glucagon. Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.


Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan. Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.


Protein-catalyzed exchange of phosphatidylinositol between rat brain microsomes and myelin. Exchange of phosphatidylinositol and phosphatidylcholine between microsomal and myelin membranes has been demonstrated. This exchange is reversible and catalyzed by soluble proteins from the brain homogenate precipitated at pH 5.1. The extent of exchange of phosphatidylinositol from microsomal membrane to myelin is dependent upon pH and temperature, with an optimum around pH 7 and at 50 degrees C. Maximum exchange was observed at approximately equal amounts of microsomal, myelin, and supernatant proteins. The extent of the catalyzed exchange increases 4- to 8-fold upon using sonicated or heat-treated myelin as an acceptor membrane. Heating of microsomal membranes results in no change. The extent of catalyzed exchange of phosphatidylcholine is less than that of the phosphatidylinositol. The exchange of other phospholipids and glycolipids between microsomal and myelin membranes cannot be demonstrated. The catalytic activity of the pH 5.1 supernatant proteins in rat brain for the exchange of phosphatidylinositol increases with age after birth and reaches a maximum around 21 days of age analogous to the process of myelination. The pH 5.1 supernatant proteins from quaking and jimpy mutant mice has normal catalytic activity.


Kinetics of the activation of human prothrombin by human coagulation factor Xa. Initial rate studies in the presence of Ca2+ and phospholipid. Steady state kinetic studies have been performed to investigate the formation of thrombin from prothrombin by human coagulation Factor Xa in the presence of Ca2+ and phospholipid. The concentration of ligand which gives 50% of the maximum velocity (K0.5) is 2.3 mM for Ca2+, 7.4 microM for phospholipid, and 0.006 microM for prothrombin. Hill plots of the Ca2+ enhancement of the reaction give a Hill coefficient of 3.1, indicating positive cooperativity. The initial velocity patterns are consistent with an ordered addition of reactants with phospholipid as the second reactant to bind to the enzyme. Although our results do not differentiate between Ca2+ or the prothrombin substrate as the first reactant to bind to Factor Xa, it is established that Ca2+ can bind to Factor Xa in the absence of the other reactants. Thus, the most probable order of addition of reactants is Ca2+, phospholipid, and the prothrombin substrate. Plots of (v)-1 versus (prothrombin)-1 or (v)-1 versus [(Ca2+)3]-1 at several constant concentrations of phospholipid indicate that the major effect of phospholipid is to increase the turnover number of Factor Xa.


Prenyltransferase. Kinetic studies of the 1'-4 coupling reaction with avian liver enzyme. Prenyltransferase catalyzes the sequential, irreversible 1'-4 condensation of isopentenyl-PP with dimethylallyl-PP and geranyl-PP to yield farnesyl-PP. A kinetic study shows substrate inhibition by isopentenyl-PP at concentrations above 0.7 microM when the concentration of geranyl-PP is 1.0 microM or less as a result of binding by the homoallylic substrate to the allylic region of the active site. Inhibition studies were carried out with the products, farnesyl-PP and PPi, and dead-end inhibitors 2-F-isopentenyl-PP and 2-F-geranyl-PP, analogues for the normal substrates. Competitive patterns were seen for farnesyl-PP and 2-F-geranyl-PP when geranyl-PP was varied, while noncompetitive patterns were found for all other combinations. A minor form of PPi, MgHPPi-, is implicated as the species of PPi in the magnesium-containing buffer which binds most tightly to the enzyme. This observation explains why K's for PPi calculated from the total concentration of PPi are much larger than K's for the organic pyrophosphates. The lack of regiospecificity in the binding of isopentenyl-PP, as evidenced by substrate inhibition patterns, introduces an element of ambiguity into mechanistic interpretations, and it is not possible to distinguish between ordered and random mechanisms on the basis of inhibition studies.


Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion. Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.


Characterization of the 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol. The 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol has been partially characterized. Sucrose density gradient sedimentation and analytical gel filtration analyses of this receptor yielded values of 3.1 S, 80,000, and 36 A for the sedimentation coefficient, molecular weight (Mr), and Stokes molecular radius (Rs), respectively. The receptor was found to be a protein by its susceptibility to protease but not nuclease digestion, and studies with N-ethylmaleimide and iodoacetamide revealed the presence of a reduced cysteine residue near the ligand binding site of the receptor. Kinetic and equilibrium binding studies showed an equilibrium dissociation constant of 7.4 x 10(-10) M (4 degrees C), an association rate constant of 1.7 x 10(7) M-1 min-1 (0 degrees C) and a dissociation rate constant of 7.2 x 10(-4) min-1 (4 degrees C, t1/2 = 16 h).


Effect of thyroid hormone on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase. Immunochemical techniques have been utilized to study the effect of thyroid status on the content and rates of synthesis and degradation of pyruvate carboxylase and pyruvate dehydrogenase in rat liver. Liver from hyperthyroid rats had twice the pyruvate carboxylase activity of normal rats while thyroidectomized rats had about two-thirds of normal activity. Pyruvate dehydrogenase complex activity was unchanged in the hyperthyroid state but was significantly reduced (by a third) in hypothyroid rats. Changes in catalytic activity during altered thyroid status were by immunochemical means to be closely related to the amount of the hepatic enzymes present. Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruvate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status. The half-life for pyruvate carboxylase was 4.6 days, and that for pyruvate dehydrogenase, 8.1 days. In both cases, the turnover time was slower than that of the average mitochondrial protein (t1/2 = 3.8 days) for the control animals.


Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages. Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.


Regulation of cholesterol synthesis in primary rat hepatocyte culture cells. Possible regulatory site at sterol demethylation. Primary rat hepatocyte culture cells were used to study the acute regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in response to 25-hydroxycholesterol, 3 beta,5 alpha,6 beta-cholestantriol, and mevalonolactone. All three effectors caused a rapid suppression of HMG-CoA reductase activity. 25-Hydroxycholesterol also caused an increase in the ratio of newly synthesized methyl sterols to newly synthesized C27-sterols. Furthermore, in 25-hydroxycholesterol-treated cells, the relative contribution of delta 24-sterol precursors to the nonsaponifiable lipid fraction increased. Di- and trimethyl-diene sterols were the dominant methyl sterols synthesized in the presence of 25-hydroxycholesterol. 3 beta,5 alpha,6 beta-Cholestrantriol (50 microM) also caused a very strong (97%) suppression of sterol demethylation; 4,4-dimethylmonoene sterols were more prominent (23%) in cells treated with 3 beta,5 alpha,6 beta-cholestrantriol, than in cells treated with 25-hydroxycholesterol (2%). The rates of both unesterified and esterified sterol synthesis increased as a function of exogenous mevalonolactone concentration. C27-sterol synthesis was saturated at a concentration of (R)-mevalonolactone which produced only a 33% suppression of HMG-CoA reductase activity. However, there was a direct relationship between the accumulation of methyl sterols and the decrease in HMG-CoA reductase activity. With the aid of triparanol, it was demonstrated that the suppression of HMG-CoA reductase activity by mevalonolactone was linked with the ability of the cells to convert squalene-2,3-epoxide into sterols. The results described in the present article support an important and perhaps necessary relationship between the rate of methyl sterol conversion of C27-sterols and the suppression or inhibition of HMG-Coa reductase in primary hepatocyte culture cells.


Inactivation of bovine pancreatic DNase by 2-nitro-5-thiocyanobenzoic acid. I. A novel inhibitor for DNase I. Though DNase does not contain any cysteine residues, incubation of the enzyme with 2-nitro-5-thiocyanobenzoic acid in the presence of Ca2+ at pH values above 7.5 results in an irreversible inactivation of the enzyme. The inactivation also occurs when Ca2+ is replaced by Mg2+, but not in their absence. Amino acid analyses after acid hydrolyses of the completely inactivated ant the native enzymes show no significant differences in composition, including tryptophan and half-cystine residues. However, sodium dodecyl sulfate gel electrophoresis indicates enzyme cleavage by the treatment with 2-nitro-5-thiocyanobenzoic acid. This reagent does not inactivate chymotrypsin and lysozyme, and under conditions where bovine DNase is inactivated, does not inactivate other nucleases such as ribonuclease, snake venom phosphodiesterase, and spleen acid DNase. However, it inactivates malt DNase and can, therefore, be considered a specific inhibitor of DNase I. The inactivation kinetics is pseudo-first order, resembling Michaelis-Menten, with an affinity constant of 16.7 mM. It is the cyano group, not the thionitrobenzoic acid of 2-nitro-5-thiocyanobenzoic acid that reacts to form cyano-DNase.


Inactivation of DNase by 2-nitro-5-thiocyanobenzoic acid. II. Serine and threonine are the sites of reaction on the DNase molecule. The amino acid compositions of various fragments isolated from DNase treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) show peptide bond cleavages to be at Thr14, Ser40, and Ser135. Isolation and characterization of radioactive tryptic and chymotryptic peptides of [14C]cyano-DNase reveal four points of peptide bond cleavage; in addition to Thr14, Ser40, and Ser135, cleavage occurs at the amino end of Ser72. Approximately 2.8 mol of [14C]cyano group are incorporated in the completely inactivated enzyme, in which 0.6 residue of Thr14, 0.8 of Ser40, and approximately 0.3 each of Ser72 and Ser135 are modified. The inactivation by NTCB can also be obtained by reacting the enzyme with a mixture of 5,5'-dithiobis(2-nitrobenzoic acid), KCN, and iodoacetate which generates NTCB. The mixture facilitates the uses of K[14C]N, which is readily incorporated into the enzyme as the [14C]cyano derivative. The reaction of NTCB with serine or threonine resembles that with cysteine.


Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells. A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.


Conformational studies of peptide heart stimulant anthopleurin A. Laser Raman, circular dichroism, fluorescence spectral studies, and Chou-Fasman calculations. Sea anemone contain a number of closely related peptide heart stimulants. In the present investigation, the conformation of anthropleurin A from Anthopleura xanthogrammica was investigated by laser Raman, circular dichroism, and fluorescence spectral methods and by the Chou-Fasman method using sequence data. The recent 13C NMR data of the peptide (Norton, R.S., and Norton, T.R. (1979) J. Biol. Chem., in press) provided useful information for the interpretation of the above-mentioned spectral data. The results from these spectral methods suggested that anthropleurin A and the related sea anemone peptides are roughly spherical in shape due to the presence of some beta-bends, possibly due to a beta-pleated sheet region and due to the 3 cystine residues in the peptide which exist in the gauche-gauche-gauche configuration. The sole tyrosine residue is exposed to the solvent, a finding which has now been confirmed by 13C NMR. The laser Raman and fluorescence spectral procedures showed that one or more of the tryptophan residues are buried. Interestingly, the reduction of the native protein with dithioerythritol did not change the spherical shape even in the presence of 5 M guanidine HCl and the carboxymethylcysteine derivative of the peptide was folded even in the presence of the denaturing agent, guanidine HCl.


The in vivo distribution of immunoreactive larger than tetrameric polyadenosine diphosphoribose in histone and non-histone protein fractions of rat liver. The macromolecular association of immunoreactive naturally occurring polyadenosine diphosphoribose n greater than 4 with histones and non-histone proteins was determined with the aid of an improved method of extraction of polyadenosine diphosphoribose and a combination of radioimmunoassay and molecular filtration. More than 99% of the naturally occurring polyadenosine diphosphoribose n greater than 4 was present in rat liver in covalent association with non-histone proteins. The chain length of the polymer varied between n = 4 and n = 34. Less than 1% of naturally occurring polyadenosine diphosphoribose n greater than 4 was almost evenly distributed between histone fractions f1, f2a, f2b, and f3. Adenosine diphosphoribose polymers of relatively long chain length were also detected in the histone fractions. The covalent association of polyadenosine diphosphoribose with non-histone proteins was demonstrated by affinity chromatography.


Random location of nucleosomes on genes for 5 S rRNA. The location of nucleosomes on genes for 5 S rRNA in rat liver was determined by the preparation of nucleosome core DNA fragments, hybridization with 5 S rRNA, RNase digestion, and gel electrophoresis. The resulting size distribution of RNA fragments was essentially the same as that found when the experiment was carried out with random DNA fragments.


Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus. An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.


Partial purification and properties of a rat brain phospholipase. At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.


Steady state and pre-steady state kinetic properties of rat liver selenium-glutathione peroxidase. The kinetic properties of partially purified rat liver selenium-glutathione peroxidase were studied under various conditions. Steady state kinetic measurements show sigmoidal saturation curves, parabolic double reciprocal plots, and Hill coefficients greater than unity. Although these kinetic results appear to show cooperative interactions between subunits, they more reflect the presence of several oxidation-reduction forms of the catalytic site. A substrate-induced transition between enzyme forms was evidence by the occurrence of a lag in the attainment of the final steady state velocity under certain preincubation conditions. This hysteretic behavior was evident only when the enzyme was incubated in the absence of reduced glutathione, the donor substrate. Thus, reduced glutathione induces the transition to the fully active form of the enzyme, a slow process requiring about 0.5 min after addition of glutathione, depending on conditions. The length, tau, of the lag period is dependent on the concentrations of enzyme and glutathione, but to a first approximation, this lag period is independent of the concentration of the hydroperoxide acceptor substrate. The lag period is also relatively independent of the nature of the hydroperoxide species. A model for the transition process that is compatible with these observations and with the possible oxidation-reduction properties of the selenium moiety of the enzyme is suggested.


Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe. Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.


Determination of the turn-off reaction for the hormone-activated adenylate cyclase. Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase.


Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles. Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.