Laser fluorometric determination of aflatoxin B1 in corn. A 2-step chromatographic separation, using both thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC), in conjunction with the high sensitivity of laser fluorometry permits extension of the detection limits of aflatoxin contamination in corn to 0.1 ppb (microgram/kg) with a 26% root mean square variation. Aflatoxin B1 is extracted from corn with water-methanol and cleaned up by TLC. The recovery of aflatoxin from the TLC plates was linear from 10 to 1000 pg. Aflatoxin B1 is converted to the more highly fluorescent B2A derivative by treatment with 1N HCl. Experiments with aflatoxin B1 standard establish a constant conversion to B2A over approximately 3 orders of magnitude in B1 concentration. An extract of the B2A aflatoxin derivative is injected onto a reverse phase HPLC column. A flowing droplet of eluant is irradiated by an amplitude-modulated 325 nm He-Cd ion laser beam, and fluorescence from the droplet is detected by a lock-in amplifier in phase with the laser modulation. Several chromatograms are presented that demonstrate the capability of this procedure for removing interfering components in the corn extract.


Thin layer chromatographic determination of citrinin. A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.


Simultaneous extraction and fractionation and thin layer chromatographic determination of 14 mycotoxins in grains. A simple, systematic analytical method for multiple mycotoxins was developed for detecting 14 mycotoxins; aflatoxins B1, B2, G1, and G2, sterigmatocystin, T-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon X, zearalenone, ochratoxin A, citrinin, luteoskyrin, and rugulosin. These mycotoxins were extracted with 20% H2SO4-4% KCl-acetonitrile (2 + 20 + 178), defatted with isooctane, and transferred to chloroform. The chloroform extract was cleaned up by silica gel column chromatography; the first 10 toxins were eluted with chloroform-methanol (97 + 3) and the remaining 4 toxins with benzene-acetone-acetic acid (75 + 20 + 5). Each fraction was analyzed by thin layer chromatography for the final determination. The method has been applied to polished rice, rough rice, corn, wheat, and peanuts as an analytical screening procedure. The detection limits in these commodities ranged from 10.00 to 800.0 microgram/kg, depending on the mycotoxin, but all limits were superior to those obtained for the individual mycotoxins by using other methods.


Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A. A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.


High pressure liquid chromatographic determination of aflatoxins in corn. A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.


Mineral oil extraction of light filth from rubbed sage: collaborative study. The official methods for extracting light filth from rubbed sage, 44.D08--44.D10, specifies a hot isopropanol pretreatment and flotation from cooled dilute isopropanol with mineral oil to extract light filth. The method gives good recoveries, but occasionally excessive interfering plant material is extracted along with the filth elements. A new method has been developed in which chloroform is used for pretreatment instead of isopropanol, and Tween 80-EDTA is added twice rather than once. This method has given consistently cleaner filter papers and better recoveries for rodent hairs, 85 vs. 79%, and for elytral squares, 95 vs. 84%. The new method has been adopted as official first action.


Extraction of light filth from canned green leafy vegetables: collaborative study. The official first action method, 44.108, for the extraction of light filth from whole or chopped canned greens specifies a water-heptane flotation that results in the flotation of an excessive amount of interfering plant material and in low recoveries. A new method has been developed using a 40% isopropanol-water-mineral oil flotation. The new method yielded a decreased amount of plant material and 84.9% recovery of the aphid spike compared with 42.5% recovery of the spike by the official method. The new method has been adopted as official first action to replace the existing method, except for broccoli.


New brine saturation technique for the extraction of light filth from rubbed sage, paprika, and corn meal: intralaboratory study. A new and widely applicable technique for the isolation of light filth from food has been developed. It includes a simple alcohol defatting followed by a brine saturation of the product by heating and cooling and a subsequent trapping of the filth elements with olive oil and tap water. The methods circumvent the use of hazardous and expensive solvents and lengthy defatting and/or pre-treatment procedures. In an intralaboratory study, recoveries of elytral squares and rodent hair fragments added to paprika, rubbed sage, and corn meal ranged from 95.8 to 100%.


Gas-liquid chromatographic determination of monosodium glutamate in soups and soup bases. A method is given for determining monosodium glutamate (MSG) in soups and soup bases by gas-liquid chromatography (GLC) of the trimethylsilyl ether derivative of glutamic acid. This method compared favorably with existing methods including GLC of the trifluoroacetate (TFA)/butyl ester derivative, and analysis on an amino acid analyzer. In addition, some samples were analyzed by GLC/mass spectrometry. The MSG content of various products ranged from approximately 0.2% in some condensed soups to 13.1% on bouillon cubes. The method, which can detect as little as 0.05%, requires only a 10 min single step derivatization at room temperature and is preferred to the TFA/butyl ester technique.


Colorimetric determination of boric acid in prawns, shrimp, and salted jelly fish by chelate extraction with 2-ethyl-1,3-hexanediol. Borate was directly chelate-extracted from foods with 5% 2-ethyl-1,3-hexanediol (EHD) in n-hexane-n-butyl acetate mixture (8 + 2), from which borate was selectively transferred into 1% NaOH, since EHD-chelated boron did not react with curcumin to develop color. Finally, an aliquot of the alkaline solution was acidified with HCl and reacted with curcumin in a rotary evaporator. Color development was increased by heating for 8 min at 80 degrees C under reduced pressure of 16 mm Hg. Frozen shrimp and prawns (peeled and with shells) and salted jelly fish were analyzed by the proposed method. Results were compared with the contemporary official method of Japan based on curcumin reaction on an incinerated sample. Over 90% of the boric acid was recovered by the proposed method when samples were fortified with 20 ppm boric acid. Recoveries were superior to those of the official method especially for shrimp and prawns with shells and salted jelly fish. Detection limit of boric acid is 1 ppm. Moreover, the method requires only about 1 hr for analysis of one sample, making it suitable for routine analysis.


Survey of some market basket commodities for polynuclear aromatic hydrocarbon content. The Food and Drug Administration multi-component regulatory procedure for determining polynuclear aromatic hydrocarbons in foods was recently used to survey 24 foodstuffs for the presence of these compounds. The procedure has a reliable limit of quantitation of 2 ppb. The potent carcinogen benzo(a)pyrene was detected in only one of the 24 products analyzed, at a level of 3 ppb. Pyrene and/or fluoranthene were found in 19 of the 24 samples examined, at levels ranging from less than 1 ppb to about 75 ppb. A comparison of the recent survey data to that obtained approximately 10 years ago reveals that the types and levels of polynuclear aromatic hydrocarbons found are essentially unchanged.


Determination of amprolium in feeds: collaborative study. The official final action method, 42.028--42.032, for determining amprolium in feeds was modified by a change in the preparation of aluminum oxide for chromatography. A premix containing 0.5% amprolium was collaboratively studied by the modified and the official methods. Compared with the modified method, 87.7% of the drug was recovered from the premix by using the official method. The modification makes possible the assay of premixes as well as finished feeds. The official final action method has been modified to incorporate this change.


High pressure liquid chromatographic determination of vitamins A and E in cereal products. A rapid method for the simultaneous determination of vitamins A and E in fortified cereal products has been developed. Saponification of retinyl or tocopheryl esters is not required, permitting direct injection of the extracted lipids onto the high pressure liquid chromatographic column without sample cleanup. Elution times of 2.46 and 3.40 min were determined for retinyl palmitate and tocopheryl acetate, respectively, using a muPorasil column and an isocratic mobile phase of hexane-chloroform (85 + 15) with a flow rate of 1.5 mL/min. The average recovery of retinyl palmitate was 99.2% (std dev. 4.28), and the average recovery of tocopheryl acetate was 94.9% (std dev. 4.10) in 2 cereals containing corn, oat, rice, and wheat. No significant amounts of naturally occurring tocopherols were found in the cereals.


Automated fluorometric determination of thiamine and riboflavin in infant formulas. Commercial infant formulas were analyzed simultaneously for thiamine and riboflavin by an automated fluorometric method and by the AOAC manual fluorometric methods. For 10 products, the mean thiamine and riboflavin results determined using the automated method ranged from 104 to 113% and 90 to 112%, respectively, of those by the AOAC manual methods. The coefficients of variation for thiamine and riboflavin ranged from 1.05 to 3.90% and 0.60 to 2.48%, respectively, for the automated methods, and 1.48 to 3.86% and 0.69 to 10.9%, respectively, for the manual methods. Using the automated method, mean recoveries of thiamine and riboflavin added to samples were 103 and 104%, respectively. The automated method used a common sample preparation to determine both thiamine and riboflavin, and gave results equivalent to, or better than, those obtained by the manual methods.


Automated fluorometric determination of vitamin C in foods. Two automated fluorometric methods were compared with the official AOAC methods for determining vitamin C in fortified ready-to-eat cereals, fruits, vegetables, baby foods, flour products, pet foods, meats, frozen dinners, juices, and nutritional health bars. Each sample was analyzed in duplicate on 2 days and included a recovery study on all products. Egberg's semiautomated method (Method 1) and Roy's automated method (Method 2) were compared with the manual AOAC tritrimetric and fluorometric methods. The correlation factor for Methods 1 and 2 were 0.999 and 0.979, respectively, when compared with the AOAC methods. The recovery study showed average recoveries of 97.8% for Method 1, 99.3% for Method 2, and 100.6% for the AOAC methods. The results suggest that Method 1 is the method of choice for the majority of the products analyzed.


Pyridine extraction method for determination of bacitracin in feeds: collaborative evaluation. Seventeen laboratories evaluated the pyridine extraction method and neomycin-sensitized agar for the determination of zinc and MD bacitracin in swine and broiler rations at 10 and 100 g/ton. The method was also applied to the analysis of 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The type of bacitracin or type of feed did not significantly affect results. The difference in results between MD and zinc bacitracin in premixes approached significance. The large coefficients of variation for premixes (ca 13%) and complete feeds (ca 15--30%) indicate operational problems. The main difficulty was evaporation of pyridine. Some laboratories were not able to evaporate it completely, whereas others lost bacitracin activity, probably due to high temperature of drying. The pyridine extraction method as in 42.200 and 42.204 should be discontinued.


Determination of chlorinated pesticide residues in foods. I. Rapid screening method for chlorinated pesticides in milk. A rapid analytical method for determining chlorinated pesticide residues in milk was developed. Thirteen pesticides were almost completely extracted. Ten mL samples of fortified milk were extracted 3 times with 20 mL portions of n-hexane as follows: (A) in the absence of water-soluble solvent; in the presence of (B) 1 mL acetonitrile; (C) 3 mL acetonitrile; (D) 5 mL acetonitrile; (E) 5 mL ethanol; (F) 5 mL acetonitrile and 1 mL ethanol. System F produced the highest pesticide recoveries but the lowest fat extraction, thus eliminating the necessity for liquid-liquid partitioning and minimizing Florisil column cleanup. Pesticide recoveries throughout the procedure were 94--103%. It was noticed, however, that the fat in high fat-containing raw milk is more readily extracted than that in commercial milk.


Determination of chlorinated pesticide residues in foods. II. Potassium permanganate oxidation for cleanup of some vegetable extracts. A potassium permanganate-dilute sulfuric acid KMnO4/dilute H2SO4 oxidation procedure was developed to supplement Florisil cleanup of some vegetable extracts. Following sample preparation and Florisil cleanup, a reaction mixture of the n-hexane eluate from the Florisil cleanup, 4% KMnO4, and 40% H2SO4 (1 + 1 + 1) was shaken in a test tube 2 min at room temperature and then centrifuged. The n-hexane phase was washed with 2 mL 0.1N NaOH and analyzed by GLC. Twelve chlorinated pesticides were completely recovered in the n-hexane phase. Aldrin was not recovered because its extreme instability caused it to decompose even in neutral solutions. Chlorinated pesticide residues in onion, garlic, carrot, and radish root were easily analyzed by the application of this oxidation procedure.


Determination of chlorinated pesticide residues in foods. II. Simultaneous analysis of chlorinated pesticide and phthalate ester residues by using AgNO3-coated Florisil column chromatography for cleanup of various samples. A simplified method suitable for simultaneous analysis of chlorinated pesticide and phthalate ester residues in various foods was developed. Chemical residues were quantitatively extracted from fatty and vegetable samples with acetonitrile as follows: Chemical standard in 0.5 mL ethanol solution was added to 10 g homogenized sample. After 3 hr, pork and beef were extracted 3 times with 20 mL portions of acetonitrile. The acetonitrile layers were diluted with water and extracted with n-hexane. Rice samples were combined with 10 mL water, 5 mL acetonitrile and 1 mL ethanol and extracted 3 times with 20 mL portions of n-hexane. The n-hexane concentrate from each sample was submitted to AgNO3-coated Florisil column chromatography. The AgNO3 coating adequately adsorbed interfering coextractives. Extracts of fish and vegetable samples were separated into 2 fractions by the above column chromatography. Supplemental cleanup procedures were also developed to accurately determine phthalate esters eluted in the second fraction. Satisfactory gas chromatograms were obtained for most samples.


Do autistic children come from upper-middle-class parents? Most studies have reported autistic children as coming from upper social economic status (SES) families. A few studies have not found any such social class bias. In order to resolve these contradictory findings, an empirical study was conducted on a statewide sample of families with autistic children. It was hypothesized that autistic children from high SES families would be associated with seven social class selection factors: (1) early age of onset, (2) early age of treatment admission, (3) normal cognitive potential, (4) complex rituals with maintenance of sameness, (5) long distance traveled for treatment, (6) limited availability of services, and (7) very detailed child history. Factors 1, 5, 6, and 7 distinguished high SES from low SES families in the predicted direction. Implications for research and treatment are discussed.


Autism and unfavorable left-right asymmetries of the brain. Utilizing computerized brain tomography, left-right morphologic asymmetries of the parietooccipital region were judged in 16 autistic patients, 44 mentally retarded patients, and 100 miscellaneous neurological patients. In 57% of the autistic patients the right parietooccipital region was wider than the left, while this pattern of cerebral asymmetry was found in only 23% of the mentally retarded patients and 25% of the neurological patients. It is suggested that unfavorable morphologic asymmetries of the brain near the posterior language zone may contribute to the difficulties autistic children experience in acquiring language.


Transformational deficits in cognition of schizophrenic children. This study examined schizophrenic children for cognitive deficits in logical thinking, symbolic imagery, and conservation. Evaluation revealed persistent failure of the aberrant subjects to achieve at the level of age expectancy. Comparison with nonpsychotic, institutionalized children demonstrated no effect due to institutionalization. By mid-teens not one of the subjects had accomplished formal operations and very few were able to master concrete operations. Implications for development and treatment were discussed.


Adult recollections of a formerly autistic child. The life history, current status, and memories of a 31-year-old man who, as a child, had been diagnosed as autistic by Kanner and others is described. Prognostic criteria as related to this individual's outcome are discussed. Some speculations about the inner life of autistic children are advanced on the basis of his recollections. Finally, some hypotheses for further research are suggested on the basis of information gained from this case review.


Surface arrays on the wall of Sporosarcina ureae. Thin sections of the cell wall of Sporosarcina ureae revealed two structurally distinct layers: a continuous amorphous zone, approximately 15 nm thick, which was adjacent to the plasma membrane, and an overlying periodic zone, approximately 16 nm thick. Sequential Triton X-100 and lysozyme treatment of isolated walls produced small fragments of the outer regular structure which allowed high-resolution, negatively stained images suitable for optical diffractometric analysis. These data suggested a tetragonal array of complex polygonal units of C-C spacing = 12 nm, with each unit joined to another by two delicate linkers. The array was entirely proteinaceous, consisting of a 150,000-dalton polypeptide which had a high affinity for Mg2+. It proved to be sensitive to chelating agents, 5 mM concentrations of Ca2+, Sr2+, or Ba2+, proteases, heat greater than or equal to 45 degrees C, sodium dodecyl sulfate, and pH greater than or equal to 5.8, but magnesium offered protection against the chelating agents and the deleterious salts.


Optimal conditions for transformation of Azotobacter vinelandii. Optimal transformation of Azotobacter vinelandii OP required a 20-min incubation of the competent cells with deoxyribonucleic acid at 30 degrees C in buffer (pH 6.0 to 8.0) containing 8 mM magnesium sulfate. Nitrogen-fixing transformants of nitrogen fixation-deficient recipients could be plated immediately on selective medium, but transformants acquiring rifampin and streptomycin resistance required preincubation in nonselective medium. The three phenotypes achieved an approximately equal and stable frequency after 17 h (six generations) of growth in nonselective medium.


Transfer of kanamycin resistance mediated by plasmid R68.45 in Paracoccus denitrificans. Plasmid R68.45 mediates the transfer of kanamycin resistance from Pseudomonas aeruginosa to Paracoccus denitrificans. Kanamycin resistance could be transferred from one strain of P. denitrificans to another, thus opening up the possibility of using R68.45 as a sex factor in P. denitrificans.


Effect of glucose on polyol metabolism by Rhizobium trifolii. The effect of glucose on polyol metabolism by Rhizobium trifolii was studied. Phenomena similar to catabolite repression and catabolite inhibition were observed. The catabolism of glucose to at least glucose 6-phosphate was required for the effects to be exerted.


Transport of C5 dicarboxylate compounds by Pseudomonas putida. Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.


Plasmenylethanolamine: growth factor for cholesterol-reducing Eubacterium. A plasmalogen, plasmenylethanolamine, is required for in vitro growth of strains of Eubacterium which convert cholesterol to coprostanol. Plasmenylethanolamine was isolated from calf brain by selective saponification of lipid fractions separated by thin-layer or column chromatography. Cholesterol-containing thioglycolate broth plus purified plasmenylethanolamine or its 2-lyso derivative supported growth of Eubacterium ATCC 21408 and a cholesterol-reducing Eubacterium isolated from baboon feces. Plasmenylethanolamine obtained from commercial sources also supported growth of these organisms, but none of a number of other pure lipids would support growth. Metabolism of the alkenyl ether group of plasmenylethanolamine occurred during growth.


Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis. A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.


Genetic mapping of toxin regulatory mutations in Vibrio cholerae. We have mapped a regulatory site mediating the hyperproduction of cholera toxin in mutants of Vibrio cholerae strain 569B. Mutations in this locus, called htx, result in the hypertoxinogenic phenotype, as measured by the ganglioside filter assay and immunoradial diffusion. Transposon-facilitated recombination was used to construct improved genetic donors in 569B parental and hypertoxinogenic mutant strains. Subsequent mapping by conjugation indicated that the htx locus was closely linked to the rif, str, and ilv loci of V. cholerae. Analysis of recombinants from these crosses suggested the following gene order: thy str htx rif ilv arg. The close genetic linkage of htx to rif (as high as 98%) resulted in a high comutation frequency of these two loci by nitrosoguanidine mutagenesis. Transfer of the htx mutant locus from a hypertoxinogenic donor to several unrelated Tox+ strains of V. cholerae caused a detectable elevation of toxin production in the recipients. These results suggest that toxin production in diverse strains of V. cholerae is controlled by a common regulatory mechanism in which the htx gene product plays a significant role.


Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon. We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes.


High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol. Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.


Minimal brain dysfunction in childhood: outcome in late adolescence and early adult years. Seventy-three patients, diagnosed in childhood as having minimal brain dysfunction syndrome and further classified as either developmental lag (38%) or organic brain syndrome (62%), were followed into late adolescence and early adult life. At follow-up 7% were free of psychiatric disorder, 80% had various types of personality disorder, and 14% were borderline psychotic. Global outcome was rated as satisfactory in 20%, unsatisfactory in 80%. Associated with an unsatisfactory outcome were these initial findings: low normal or borderline intelligence, multiplicity of behavioral and neuropsychological findings, learning disabilities, special class placement, and initial classification of organic brain syndrome. Initial differentiation between developmental lag and organic brain syndrome foreshadowed different psychopathological features having significant polarities and little overlap, as well as different global outcome.


Differential diagnosis of fugue-like states. Fugue-like states, though usually considered hysterical or epileptic in origin, can occur with many other psychiatric and neurologic conditions. The survey of literature should help the clinician with differential diagnosis. Nine illustrative cases are described and guidelines for investigating a case of fugue are outlined.


The clinical utility of reproduction drawing tests with low IQ patients. Bedside mental status testing in low IQ, poorly educated patients is often difficult. These patients, who comprise a significant proportion of the population served by major inner city public hospitals, have a limited ability to perform adequately on many higher cortical function tests. Because of this, it is often difficult to accurately differentiate between an organic brain syndrome and the effect of depressed intelligence in these patients. Copying line drawings and other constructional tasks are a very important element in a complete mental status examination, as they are frequently failed by patients with organic brain disease. The applicability of drawing tasks in the low IQ patient is an important issue; this study demonstrates that selected drawings can be effectively utilized to identify brain dysfunction in this low IQ population.


Cognitive impairment and treatment outcome with alcoholics: preliminary findings. The relationship of cognitive impairment to treatment outcome in a sample of male alcoholics (N = 30) was explored. The Brain-Age Quotient (BAQ), an index of problem solving and adaptive abilities based on a battery of neuropsychological tests, was used as the summary measure of cognitive impairment. High BAQ scores were found to be more highly related to clinical ratings of patients' level of functioning on the treatment ward, to the successful completion of inpatient treatment, and to fewer relapses, longer abstinence periods, and lower rates of alcohol consumption at a 1 year follow-up than were lower BAQ scores. The implications of these findings with regard to appropriate treatment planning are discussed.


Effects of divalent cations on thermophilic inorganic pyrophosphatase. Divalent cations were shown to affect the structure and thermostability of thermophilic inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] purified from Bacillus stearothermophilus and thermophilic bacterium PS-3. The properties of the enzymes from the two sources were found to be very similar. The enzymes were very unstable to heart in the absence of divalent cations, being inactivated gradually even at 40 degrees C. However, they became stable to heat denaturation in the presence of Mg2+, between pH 7.8 and 9.0. Similar induced thermostability was detected when Mn2+, Co2+, Ca2+, Cd2+, and ZN2+ were added, though the latter three cations were not essential for enzyme activity. On adding divalent cations, the optical properties such as absorption spectra, fluorescence spectra, and circular dichroism (CD) were changed. Gel filtration and disc electrophoresis revealed that the molecular weight of both enzymes was 5.4 x 10(4) in Tris-SO4 buffer and 11 x 10(4) in Tris-HCL buffer, suggesting monomer-dimer transformation. In the presence of divalent cations in Tris-SO4 fuffer, the enzymes dimerized; this was confirmed by sedimentation velocity measurements. The enzymes in Tris-HCL buffer did not show thermostability unless divalent cations were added. The results in the present study indicate that binding of divalent cations to each enzyme caused some conformational change in the vicinity of aromatic amino acid residues leading to dimerization of the enzyme molecule so that it became thermostable. It was also suggested that histidyl residues play an important role in the thermostability induced by divalent cations on the basis of the pH dependencies of thermostability and CD spectra.


The site of inhibition of cell wall synthesis by 3-amino-3-deoxy-D-glucose in Staphylococcus aureus. The inhibition of growth and cell wall synthesis by 3-amino-3-deoxy-D-glucose (3-AG), which is known to be one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., was almost completely overcome by glucosamine and N-acetylglucosamine in Staphylococcus aureus but scarcely affected by D-glucose and D-fructose. The antibiotic did not inhibit the incorporation of [14C]glucosamine and [3H]N-acetylglucosamine into the acid-insoluble fraction, but rather enhanced the incorporation of [14C]glucosamine. On the other hand, it inhibited the incorporation of D-[14C]fructose into the cell wall fraction but hardly affected the incorporation of D-[14C]fructose into the acid-insoluble fraction in the presence of pencillin G. Based on these results, it is suggested that the site of primary action of 3-AG is the formation of glucosamine-6-phosphate from D-fructose-6-phosphate, which is catalyzed by glucosamine synthetase [EC 2.6.1.16].


Formation of roseoflavin from guanine through riboflavin. A synthesis of roseoflavin by Streptomyces davawensis from guanine through riboflavin was demonstrated. The lines of evidence are (1)incorporations of 14C of [2-and U-14C] guanine and [2-14C] riboflavin into roseoflavin, (2) no incorporation of 14C of [8-14C] guanine into roseoflavin, (3) localizations of 14C in roseoflavin, and (4) a decrease of specific radioactivity of roseoflavin formed from [2-14C]guanine on addition of riboflavin to the culture. The 14C atoms in roseoflavin formed were localized by radioactivity analysis of the NaOH-hydrolysis products, i.e., urea and 1,2-dihydro-6-methyl-7-dimethylamino-2-keto-1-D-ribityl-3-quinox-alinecarboxylic acid (QC), a new substance. These hydrolysis products were identified by the isolation of dixanthylures, decomposition with urease, and from  the properties of QC and QC tetraacetate isolated. These finding suggest that the pyrimidine ring of guanine is conserved in the formation of roseoflavin from guanine through riboflavin.


Two components of chorismate mutase in Brevibacterium flavum. Chorismate mutase of Brevibacterium flavum, a common enzyme in phenylalanine and tyrosine biosynthesis, was separted into two different component, A and B, with molecular weights of 250,000 and 25,000, respectively, by ammonium sulfate fractionation or gel-filtration. Both components were essential for the enzymatic activity. In the presence of the reaction substrate, chorismate, the two components associated reversibly to give an active enzyme complex with a molecular weight of 320,000. Binding sites of the feedback inhibitors, phenylalanine and tyrosine, on the enzyme were localized on component A as determined by hybridization experiments with the wild-type and mutant components. Tyrosine repressed the synthesis of component B much more strongly than that of component A, while phenylalanine did not show any significant repressive effect on either component. The wild-type strain No. 2247 had four times more component A than component B. Elution patterns in gel, DEAE-cellulose or hydroxyapatite column chromatography as well as the disc-gel electrophoretic pattern of chorismate mutase component A and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase activities completely overlapped, suggesting the presence of a bifunctional protein having the two activities. In accord with this suggestion, chorismate mutase as well as DAHP synthetase was insensitive to feedback inhibition by phenylalanine and tyrosine in all the 3-fluorophenylalanine-resistant mutants tested that excreted both phenylalanine and tyrosine. All the phenylalanine and tyrosine double auxotrophs defective in chorismate mutase lacked component B but not A.


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation. The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid  undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.


Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver. The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.


Process of iodination of thyroglobulin and its maturation. II. Properties and distribution of thyroglobulin labeled in vitro or in vivo with radioiodine, 3H-tyrosine, or 3H-galactose in rat thyroid glands. With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.


Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex. Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.


Cerebroside sulfotransferase activity in human lung tissues. An elevated level in lung adenocarcinoma. Cerebroside sulfotransferase activity was demonstrated in particulate fractions from human lung and its carcinoma tissues. The activity in human lung adenocarcinoma was significantly higher than those in a different histological type of carcinoma (squamous cell carcinoma) and in normal tissue from which each carcinoma was derived.


Crystallographic data for cytochrome c3 from two strains of Desulfovibrio vulgaris, Miyazaki. Two crystalline forms of cytochrome c3 isolated from two strains of Desulfovibrio vulgaris, Miyazaki, tentatively designated as D. vulgaris, Miyazki F and  D. vulgaris, Miyazaki K, have been found. Both belong to the orthorhombic system, space group P2(1)2(1)2(1), but have different cell dimensions; a=54.1, b=68.9 and c=35.0 A for D. vulgaris, Miyazaki F, and a=43.5, b=41.2, and c=62.9 A for D. vulgaris, Miyazaki K. The asymmetric unit of each crystal contains one molecule of cytochrome c3.


Catalytic activity of the chromophore of desulfoviridin, sirohydrochlorin, in sulfite reduction in the presence of iron. Sirohydrochlorin chromophore prepared by acetone/HCl treatment of desulfoviridin, the sulfite reductase from Desulfovibrio, catalyzed the reduction of sulfite to sulfide and thiosulfate in equimolar amounts when coupled with a hydrogen-hydrogenase-methyl viologen system. This activity was manifested at acidic pH and increased exponentially with decrease in pH. The Km value for sulfite was nearly 10 times that of desulfoviridin. Inorganic iron was necessary for the reduction, since inactivation occurred on passage through a Sephadex LH-20 column or in the presence of 2,2'-bipyridine, and reactivation was observed on adding iron. The chromophore catalyzed the reduction of dithionite and hyroxylamine.


On the formation of gamma-aminobutyric acid from putrescine in brain. Gamma-aminobutyric acid is not formed in significant amounts from putrescine by incubation with rat brain homogenates. However, it is formed if acetyl-CoA is added to the incubation medium. This is taken as further evidence for the existence of a metabolic pathway in mammalian brain which comprises acetylation of putrescine to monoacetyl putrescine and oxidative deamination of monoacetyl putrescine by MAO. Nerve cells and glia cells have comparable capacities for putrescine degradation along this pathway.


Carboxymethylation of a minor ribonuclease from Aspergillus saitoi. (1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.


Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus. Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.


Isolation and amino acid sequences of proline-rich peptides of human whole saliva. Three basic peptides with extremely high proline contents were isolated from human whole saliva. The amino acid sequences of two of these proline-rich peptides comprising 57 and 38 residues were determined by conventional methods. The sequence suggested that the smaller peptide was derived from the larger one and also revealed the occurrence of characteristic repeating units within the molecules. The present study is the first to describe this structural feature of proline-rich proteins or peptides.


A determination of H2O2 release by the treatment of human blood polymorphonuclear leukocytes with myristate. Free H2O2 released from human blood leukocytes during phagocytosis into the extracellular medium was highly reactive with the ferric form of HRP, forming an enzyme-substrate complex which was identical to HRP-H2O2 compound II. The formation of HRP-H2O2 compound II was employed for assaying the rates of H2O2 release by leukocytes upon addition of bacteria or myristate. The treatment of normal human blood leukocytes with myristate resulted in a marked stimulation of H2O2 release compared to phagocytizing cells. The activity of H2O2 release in response to myristate was found to be deficient in the leukocytes of two patients with chronic granulomatous disease. This assay method with myristate supplementation is so sensitive and specific that it should be useful for the diagnosis of chronic granulomatous disease.


Substrate specificity and reaction mechanism of putrescine oxidase. Putrescine oxidase [EC 1.4.3.4] of Micrococcus rubens oxidizes many kinds of synthetic polyamines: triamines (spermidine types), tetramines (spermine types), and N-substituted putrescines. Polyamines possessing terminal 4-aminobutylimino groups in their structures were more active as substrates. Putreanine was oxidized at a rate comparable to that of putrescine, and was converted to 1-pyrroline and beta=alanine. Activities and Km values for polyamines were affected by the substituent attached to the 4-aminobutylimino group of the polyamine, and especially by its methylene chain length. It was also found that two types of oxidation occurred in the oxidation of polyamines by putrescine oxidase. When the moieties attached to the 4-aminobutylimino groups in polyamines were less hydrophobic, these polyamines were oxidized at the secondary amino groups to form 1-pyrroline. Polyamines which contained a hydrophobic substituent attached to the 4-aminobutylimino moiety to form ammonia. N,N'-Bis (4-aminobutyl)-1,3-diaminopropane ([II, 4-3-4]) and N-(4-aminobutyl)-N'-(3-aminopropyl)-1,3-diaminopropane ([II, 4-3-3]) were oxidized to form 1-pyrrolinium salt derivatives as a result of oxidation of the terminal primary amino groups. It was concluded that the essential structure for substrates of putrescine oxidase is a 4-aminobutylimino group (NH2(CH2)4NH-).


Occurrence of free ceramides in Bacteroides fragilis NCTC 9343. The occurrence of free ceramides at high concentrations was demonstrated in the chloroform-methanol extractable lipids of Bacteroides fragilis NCTC 9343. The long-chain bases were isolated from the free ceramides and identified as branched and normal saturated dihydroxy bases with carbon chains consisting of 17, 18, and 19 atoms. The major fatty acid was 3-hydroxy 15-methylhexadecanoic acid. The major molecular species of the ceramides were identified by gas chromatography-mass spectrometry and gas chromatography of the cleaved products as LCB-d-iso17: 0-3-OH iso17: 0 FA, LCB-d-anteiso17: 0-3-OH iso17: 0 FA, LCB-d-iso18: 0-3-OH iso17: 0 FA, and LCB-d-anteiso19: 0-3-OH iso17: 0 FA.


Characteristics of D-leucine uptake by mouse Ehrlich ascites tumor cells. In a previous paper, we reported that various D-amino acids were taken up several times more effectively than the corresponding L-isomers into several tumors tested in vivo. In order to investigate this further, the in vitro uptake of D-[14C]leucine by Ehrlich ascites tumor cells was investigated in comparison with that of L-[14C]leucine. The distribution ratio, the effects of amino acids and pH, and an approximately linear Lineweaver-Burk plot indicated that D-leucine was transported by an active transport system for L-leucine. Vmax and ku, the first-order rate constant for the unsaturable component, for the uptake of D- and L-leucines decreased with a fall in temperature. The activation energies for Vmax and ku were in the range of 5-10 and 18-21 kcal/mol, respectively. The values for L-leucine were greater than those for D-leucine. Km for D-leucine transport increased with decreasing temperature, whereas Km for L-leucine decreased. This difference suggests that the large alkyl chains of D- and L-leucines bind to different portions of a carrier protein.


Age-dependent constitution of chondroitin sulfate isomers in cartilage proteoglycans under associative conditions. Associated proteoglycans were prepared with guanidine-HCl from bovine articular cartilage of various ages. They were purified and fractionated by equilibrium centrifugation in cesium chloride (CsCl) density gradients. The compositions of chondroitin sulfate (CS) isomers in associated proteoglycans of articular cartilages of three ages were compared based on the relative amounts of disaccharide units. The results indicated that the proportions of 4-sulfated disaccharide units comprised around 2/3, 1/3, and 1/6 of the total CS in the associated proteoglycans of calf, 18-month-old cow, and 8-year-old cow, respectively. In contrast, the proportions of 6-sulfated disaccharide units in the proteoglycans were in the reverse order; they comprised nearly 1/3, 1/2, and 2/3 of the total CS, respectively, at the three ages. Thus, with increasing age, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units decreased significantly. With the decrease of CsCl density in the gradients, the proportion of 4-sulfated disaccharide units to total CS as well as that of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased in the associated proteoglycans of all ages. The increased ratios of 4-sulfated to 6-sulfated disaccharide units with decreasing CsCl density were significant among the individual proteoglycans: 1.84-2.36 in calf, 0.40-0.89 in 18-month-old cow, and 0.16-0.28 in 8-year-old cow.


In vivo degradation of rat globin messenger RNA during maturation of reticulocytes. The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.


Separation of the alpha chains of type I and III collagens by SDS-polyacrylamide gel electrophoresis. A method for the separation of type III collagen from type I collagen by SDS-polyacrylamide gel electrophoresis has been developed. This is based on the observation that the presence of 3-4 M urea decreases the mobility of the alpha 1 [III] chain to a greater extent than those of the alpha 1[I] and alpha 2 chains, although the alpha 1[I] and alpha 1[III] chains move at the same rate in the absence of urea. An attempt to separate the alpha 1[II] chain of type II collagen from the alpha 1[I] chain was unsuccessful under the experimental conditions employed.


Molecular arrangement of troponin-T in the thin filament. 1. Chymotrypsin cleaved troponin-T of skeletal muscle into two subfragments, i.e., troponin-T1 and -T2, each of which could be isolated by the use of DEAE-Sephadex. Troponin-T1 was a single subfragment with a molecular weight of 26,000 (chicken) or 22,000 (rabbit) daltons. Troponin-T2 consisted of two subfragments with molecular weights of about 13,000 daltons. Results obtained indicated that the smaller subfragment was formed by digestion of the larger subfragment of troponin-T2. 2. Antibodies against troponin-T1 and -T2 formed regular transverse striations along the whole length of thin filaments with 38 nm intervals, as was found reviously using antibodies against whole troponin complex as well as troponin components (Ohtsuki, I. et al., 1967; Ohtsuki, I. 1974 and 1975). 3. The first anti-troponin-T1 striation was situated 40 nm from the top of the filament. The first anti-troponin-T2 striation was 27 nm from the filament top and coincided with the first striations formed by antibodies against troponin-C or -I. 4. Troponin-T1 and the larger subfragment of troponin-T2 bound to tropomyosin which had been coupled to Sepharose, whereas the smaller subfragment of troponin-T2 did not.


Factors affecting the transient tension change after applying stepwise length change to glycerol-treated muscle fibers. Effects of temperature, divalent cations, and modification with p-chloromercuribenzoate. The dynamic properties of cross-bridge movement were investigated in glycerol-treated muscle fibers under various conditions by analyzing tension responses to two types of length change. First, the fiber bundles were stretched linearly with time for 0.3 s from the rest length (L0) by 2.5% of L0, suddenly released, then fixed at L0 (sudden release of the slow stretch). Second, they were stretched for 0.01 s by 2.5% of L0, then held at the plateau length (a quick stretch). 1. The transient tension responses following both length changes were divided into three phases: (i) very quick recovery of tension (0 approximately 0.05 s), (ii) quick recovery (0.05 approximately 0.3-0.4 s), and (iii) gradual recovery (0.3-0.4 s approximately several seconds). 2. The effects of activating conditions on the rates of the quick phases (0 approximately 0.3-0.4 s) were not associated with those on the nucleoside triphosphatase [EC 3.6.1.3] rates: the rates of the quick phases increased with increase in temperature and Mg2+-ATP concentration, with decrease in Ca2+ concentration, and also on replacement of Mg2+-ATP by Mg2+-ITP or Mn2+-ATP. Only a small amount of ADP, 0.07 mol per mol of myosin (Fig. 24 in the preceding paper), was liberated during the quick recovery phases. 3. The remaining slow tension recovery was concluded to be associated with one cycle of ATP splitting, and progressed very smoothly. This suggests that most of the cross-bridges do not exist in a synchronously dissociated state during one cycle of ATP splitting.


Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease. The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.


Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction. The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.


Studies of a calcium-activated neutral protease from chicken skeletal muscle. II. Substrate specificity. Calcium-activated neutral protease purified from chicken skeletal muscle hydrolyzed myofibrillar proteins, tubulin, spectrin, and oxidized insulin B chain, but hardly hydrolyzed synthetic or natural peptides.


The association between major sialoglycoprotein and spectrin of human erythrocyte membranes as detected by fluorescence resonance energy transfer. The interactions among the major sialoglycoprotein and peripheral proteins of human erythrocyte membranes were investigated by the long range resonance energy transfer between different fluorescent moieties separately conjugated to proteins. Consequently, direct association between the major sialoglycoprotein and spectrin was observed and divalent cations were required for their association.


Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes. Application to ribonuclease A. The 270 MHZ proton MNR spectra of an aqueous solution of ribonuclease A in the presence of hexacyanochromate ions were measured. The C2 and C4 proton signals of the His 12 and His 119 residues were selectively broadened, indicating that this ion was bound to the active site. From analyses of concentration dependences of diamagnetic shifts, one hexacyanocobaltate ion was found to bind to the active site, with the dissociation constant of 8 mM. The paramagnetic hexacyanochromate ion is useful for mapping the active sites of enzymes for anionic substrates.


Purification of immunologically active HLA-A and -B antigens by a series of monoclonal antibody columns. Serologically and biochemically pure preparations of detergent and papain-solubilized HLA-A2 and HLA-B7 antigens were isolated by high pH elution from a series of immunoaffinity columns constructed from monoclonal antibodies with specificity for HLA-A2 and HLA-B7 antigens. These preparations retained the immunological activity and quaternary structure of the native molecule and should provide suitable reagents for insertion into liposomes and probing the role of major histocompatibility antigens in lymphocyte interactions.


Purification of the H-2Kk molecule of the murine major histocompatibility complex. The intact H-2Kk antigen has been detergent-solubilized and purified using an immunoabsorbent column prepared from the 11-4.1 monoclonal antibody described by Oi et al. (Oi, V. T., Jones, P. P., Goding, J. Current Topics in Microbiology and Immunology (Melchers, F., Potter, M., and Warner, N. L., eds) Vol. 81, pp. 115-129, Springer-Verlag, New York). The mild conditions used for elution from the column, 0.5% deoxycholate in 10 mM Tris buffer, pH 8, with 0.14 M NaCl, result in recovery of 70 to 100% of the allogeneic serological activity. A murine lymphoma, RDM-4, was found to express high levels of H2-Kk; approximately 2 X 10(6) molecules/cell. Milligram quantities of H-2Kk can be purified readily using these cells.


A new polyamine, thermospermine, 1,12-diamino-4,8-diazadodecane, from an extreme thermophile. A new polyamine has been extracted from an extreme thermophile, Thermus thermophilus, and its chemical structure was determined as 1,12-diamino-4,8-diazadodecane, NH2(CH2)3NH(CH2)3NH(CH2)4NH2, based on its proton NMR, 13C NMR, and mass spectra. A trivial name "thermospermine" is proposed for the new compound.


Dissociation of thrombin from platelets by hirudin. Evidence for receptor processing. Hirudin inhibited the binding of human 125I-alpha-thrombin to the saturable, but not the nonsaturable, sites on washed human platelets. When hirudin was added to a thrombin-platelet mixture, it caused a biphasic dissociation of bound thrombin. A partial dissociation was too rapid to measure and was followed by complete dissociation with a first order rate constant of about 10(-2) s-1. The fraction of bound thrombin in the more slowly dissociable form increased from essentially none after a 5-s preincubation of thrombin and platelets to as much as 75% of saturable binding after a 4-min preincubation. Transition to the slowly dissociable state was not accompanied by an increase in the amount bound and was not observed with active site serine-derivatized thrombin. This is the first evidence with intact platelets of a binding characteristic that depends, as does platelet stimulation, on catalytically active thrombin, suggesting that it may represent physiologically significant receptor processing.


Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria. Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.


Hybrid formation for liganded hemoglobins A and C at subzero temperatures. The kinetics of formation of the asymmetric carbonmonoxyhemoglobin hybrid (alpha beta)A(alpha beta)C from the parent molecules alpha 2 beta 2A and alpha 2 beta 2C have been studied by electrophoresis at subzero temperatures (down to -40 degrees C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH greater than 10, t = -40 degrees C, the hybrid between alpha 2 beta 2A and alpha 2 beta 2C is stable for several hours. A rapid thermal quenching of a mixture of alpha 2 beta 2A and alpha 2 beta 2C prevented hybrid formation during the time required to separate the 2 molecules.


Carbohydrate of the human plasminogen variants. III. Structure of the O-glycosidically linked oligosaccharide unit. The preceding two manuscripts in this issue (Hayes, M. L., and Castellino, F. J. (1979) J. Biol. Chem. 254, 8768-8771, 8772-8776) describe the isolation and characterization of glycopeptides from human plasminogen affinity chromatography variants 1 and 2. Plasminogen variant 1 contains an asparagine288-based branched carbohydrate structure, which has been established in the immediately preceding manuscript. This structure is absent in variant 2. Plasminogen variants 1 and 2 contain a threonine-based glycoconjugate. This latter structure has been established by combination of methylation data, glycosidase digestions, periodate oxidations, and Smith degradations of the beta-eliminated reduced oligosaccharides. One glycopeptide unit, isolated from both plasminogen variants 1 (1D) and 2 (2D) possessed the following structure: Sia alpha 2 yields 3Gal beta 1 yields 3GalNAc-Thr. The threonine was found to be residue 345 in the Glu-plasminogen sequence. Another glycopeptide unit was also found to be present, in lower yields in both variants 1 (1E) and 2 (2E). The structure of this unit was: Sia alpha 2 yields 3Gal beta1 yields 3GalNAc-Thr. : formula: (see text), alpha 2,6 Sia. Again Thr 345 was the glycosylated amino acid. The amino acid sequence around the glycosylated threonine was found to be NH2-Ala.Pro.Thr(CHO).Ala.Pro.Pro.Glu.


The sites of the lactoperoxidase-catalyzed iodination of human fibrinogen. A study of those tyrosines in fibrinogen which are surface-oriented and which may be involved in polymerization has been investigated using as a probe iodination catalyzed by lactoperoxidase. The iodine distribution in the major cyanogen bromide fragments was studied. A fragment of the B beta chain extending beyond residue 118 had the highest specific activity. Tyrosine 119 was identified as the residue most susceptible to iodination. There was no difference in susceptibility to iodination of N-DSK (A alpha 1-51, B beta 1-118, gamma 1-78)2, Ho1-DSK (first hydrophobic disulfide knot), and Hi2-DSK (second hydrophobic disulfide knot). Tyrosines 18 and 32 of the gamma chain of N-DSK were not significantly iodinated in fibrinogen, but tyrosines 1 and 68 were labeled, as was the tyrosine of the A alpha chain. The data indicate that there are regions of the hydrophobic disulfide knot, Ho1-DSK, which are surface-oriented. The distribution of iodine as mono- and diiodotyrosine in N-DSK and Ho1-DSK reflected the percentage (83 and 17, respectively) found in iodinated fibrinogen from which these fragments were prepared. In contrast the segments of the B beta chain extending beyond Met118 contained 46% of the iodine in diiodotyrosine, while the A alpha chain fragment, Hi2-DSK, contained 28% as diiodotyrosine. No significant iodination of histidine was detected.


Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ. A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.


Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides. Asparagine-linked oligosaccharides of glycoproteins undergo extensive modification or "processing" following their attachment to protein. A key step in post-glycosylation processing is the sequential removal of glucose residues from the protein-linked oligosaccharide. We have studied rat liver preparations which catalyze removal of glucose from Glc3Man9GlcNAc, Glc2Man9GlcNAc, and Glc1Man9GlcNAc. Detergent solubilization studies, inhibitor studies, and temperature-activity profiles indicate that at least two distinct glucosidases are present in the membranes. One of these glucosidases removes the distal glucose from Glc3Man9GlcNAc, and the other glucosidase sequentially removes glucose from Glc2Man9GlcNAc and Glc1Man9GlcNAc. The latter glucosidase has been solubilized from the microsomal memrbranes and purified 12-fold. The glucosidases, which are integral membrane proteins, are localized in the rough and smooth microsomes and appear to be located on the cisternal surface of the microsomal vesicles. These glucosidases are suggested to be of biological importance in catalyzing the initial events in the post-glycosylation processing of cellular glycoprotein.


Syncatalytic modification of chicken liver xanthine dehydrogenase by hydrogen peroxide. The nature of the reaction. Xanthine dehydrogenase engaged in catalyzing the oxidation of substrate by oxygen is repidly inactivated by the hydrogen peroxide generated during the reaction. Experimental evidence shows that peroxide reacts more readily with the reduced than with the oxidized form of the enzyme. Inactivation results from modification of the cyanolysable sulfur present at the molybdenum center.


Interfacial structure and lipase action. Characterization of taurodeoxycholate-didecanoylglycerol monolayers by physical and kinetic methods. Surface pressure-area isotherms for 1,3-didecanoyl-glycerol (dicaprin) were determined as a function of the concentration of taurodeoxycholate in the subphase. Analysis of these curves indicates that, from 0.05 to 0.80 mM bile salt, surface structure is dependent only on the surface concentration of the diglyceride. The limiting areas for dicaprin in the presence and absence of bile salt were about 38 A2/molecule. Subjecting the monolayers to hydrolysis by pancreatic lipase yielded kinetic data which, together with the physical studies, support a model for monolayer glyceride molecules undergoing discrete changes of state. In the absence of bile salt, the relatively expanded state exhibits an area of 75 A2/diglyceride molecule and is not a substrate for pancreatic lipase B. The more condensed state exhibits an area of 38 A2/diglyceride molecule and is hydrolyzed at a rate proportional to its concentration in the monolayer. Taurodeoxybholate at 0.05 to 0.60 mM shifts the apparent area of the expanded state to 360 A2/diglyceride molecule.


Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. Rat liver lysosomal preparations incubated with 10(-5) M L-[4,5-3H]leucine methyl ester hydrolyzed the methyl ester and accumulated radioactivity within a particulate compartment. The acculated radioactivity was identified as free leucine by thin layer chromatography. Free leucine was not itself taken up by the lysosomal preparations. The capacity to accumulate leucine was identified as a specific property of lysosomes and was thought to result from the trapping of the free amino acid within the lysosome following the hydrolysis of the methyl ester. Lysosomes also accumulated phenylalanine, serine, and alanine when incubated with the corresponding methyl esters. Leucine accumulation was inhibited by submillimolar concentrations of chloroquine, by the protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone, and by lowering the pH below 7.0. Efflux of leucine from the lysosomes was highly temperature dependent (activation energy 33 kcal/mol). No evidence was found to suggest that leucine efflux was a carrier-mediated process. The results provide a new methodology for the study of amino acid movements across lysosomal membranes.


Reversible uncoupling of photophosphorylation by a new bifunctional maleimide. A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.


Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation. Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.


Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl. Adenylate deaminase from rat skeletal muscle has been studied with the objective of understanding how the activity of the enzyme is regulated in vivo. ATP and GTP inhibit the enzyme at low concentrations in the presence of 150 mM KCl. The ATP inhibition is reversed as the ATP concentration is raised to physiological levels. The GTP inhibition is reversed as the GTP concentration is raised to unphysiologically high levels. In the presence of physiological concentrations of ATP, the GTP inhibition is also greatly diminished, but inhibition by orthophosphate remains strong. The apparent affinities of the enzyme for GTP, ATP, and orthophosphate are reduced as the pH is decreased from 7.0 to 6.2. ADP also reduces the apparent affinities of the enzyme for the inhibitors. The regulatory effects of GTP, ATP, and ADP are produced primarily by their unchelated forms. Comparison of the kinetic behavior of the enzyme in vitro with metabolite concentrations in vivo indicates that the major variables that regulate the activity of adenylate deaminase of muscle in vivo are the concentrations of AMP, ADP, orthophosphate, and H+.


The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes. In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.


Proton NMR study of methemoglobin and its isolated chains. Effect of the subunit association on the structure of the subunits. In order to investigate the effect of the alpha beta subunit contacts on the subunit structure of human adult methemoglobin, the hyperfine shifted proton NMR spectra of several high spin complexes (water, cyanate, thiocyanate, formate, fluoride, and nitrite) and low spin complexes (imisazole, azide, and cyanide) of hemoglobin and its isolated subunits were characterized at 220 MHz and 22 degrees C. The spectra of ferric low spin derivatives of the isolated subunits were approximately superimposable on the corresponding hemoglobin spectra. On the other hand, the high spin spectra of the isolated subunits were greatly different from each other. The spectral anomaly in the ferric high spin complexes of the isolated beta subunit were interpreted to indicate other structural change than the hemichrome formation in the beta heme pocket. Difference in the subunit association effect between the high and low spin complexes of the isolated beta subunit was interpreted on the basis of a conformational change of the apoprotein dependent on the spin state of the beta heme iron.


Formation of stable anhydrides from CoA transferase and hydroxamic acids. Acetohydroxamic acid reacts with the enzyme-CoA form of succinyl-CoA:3-ketoacid coenzyme A transferase to give an inactive product with a rate constant of 860 M-1 min-1 at pH 8.1, 25 degrees C. The reaction is reversible in the presence of coenzyme A and has an equilibrium constant of 0.040. The product is an anhydride that is an analog of the intermediate that has been postulated in the normal catalytic pathway; it is inactive because coenzyme A does not react with the acyl group of the hydroxamic acid. The equilibrium constant for formation of the anhydride from the thil ester of enzyme and methyl 3-mercaptopropionate is 75 times larger than the equilibrium constant of 2.2 for the formation of N,O-diacetylhydroxylamine from acetohydroxamic acid and acetyl-CoA. This shows that the enzyme stabilizes the anhydride at the active site by at least -2.6 kcal mol-1. Succinomonohydroxamic acid reacts with enzyme-CoA as both a substrate and an inactivator, with relative rate constants of 25:1. The inactivation is irreversible, indicating that the enzyme provides a larger stabilization of at least -5.9 kcal mol-1 for the anhydride of an analog of the specific substrate, succinate. The results are consistent with the hypothesis that the enzyme stabilizes an anhydride that is formed at the active site during turnover of normal substrates through a stepwise reaction mechanism.


Enzymic memory. Steady state kinetic and physical studies with ascorbate oxidase and aspartate aminotransferase. Enzymic memory is a kinetic phenomenon observable in double displacement mechanisms. The defining feature of enzymic memory is the occurrence of different rates of transfer for a common transferable group from the substituted enzymes obtained with different donor substrates. Memory behavior was previously demonstrated for both the bovine and human liver rhodaneses (EC 2.8.1.1). Steady state kinetic tests for enzymic memory have now been done with ascorbate oxidase (EC 1.10.3.3) and aspartate aminotransferase (EC 2.6.1.1). The results were positive with ascorbate oxidase, which showed an oxygen reactivity ratio of 1:20:300 for the reduced enzymes obtained with reductate, araboascorbate, and ascorbate, respectively. Results were negative for the aminotransferase tested with the alternate donors glutamate and cysteine sulfinate, with oxaloacetate as the common acceptor. The structural basis of the ascorbate oxidase results was probed by comparison of both the ultraviolet absorption and fluorescence spectra of the oxidized enzyme with those of the reduced forms obtained with ascorbate and reductate. The results are consistent with a conformational basis for the memory phenomenon.


A concanavalin A-resistant Chinese hamster ovary cell line is deficient in the synthesis of [3H]glucosyl oligosaccharide-lipid. In this report we present an initial determination of the biochemical defect present in a Chinese hamster ovary cell line selected for resistance to concanavalin A. Membranes of this mutant, B211, incorporated at least 10-fold less mannose from GDP-[14C]mannose into oligosaccharide-lipid than membranes of the wild type. In the presence of dolichol phosphate, membranes of the mutant and wild type exhibited similar rates of synthesis of number of early intermediates, namely, mannosylphosphoryldolichol, N-acetylglucosaminyl- and N,N'-diacetylchitobiosylpyrophosphoryldolichol, glucosylphosphoryldolichol, and mannosyloligosaccharide-lipid. The membranes of B211 did not incorporate glucose from UDP-[3H]glucose into oligosaccharide-lipid or protein. Comparison by gel filtration chromatography of oligosaccharides derived from the oligosaccharide-lipids of B211 and wild type cells labeled with [2-3H]mannose revealed that B211 cells incorporated little if any label into an oligosaccharide corresponding to the most excluded oligosaccharide labeled by wild type cells. This concanavalin A-resistant cell line appears to lack the ability to glucosylate oligosaccharide-lipid.


Correlation of ultrastructure of reconstituted sarcoplasmic reticulum membrane vesicles with variation in phospholipid to protein ratio. We have previously described the reconstitution of functional membrane vesicles with lipid content similar to that of the normal sarcoplasmic reticulum membrane (approximately 1.0 mumol of phospholipid/mg of protein). The present study describes methodology to prepare reconstituted membrane vesicles with defined phospholipid to protein ratio, both lower and higher than that of the original membrane. The Ca2+ loading rate and efficiency are greatest in the membranes of highest protein content (0.38 mumol of phospholipid/mg of protein), decline slowly as the lipid content is quadrupled, and decrease markedly as the lipid content is quadrupled again. Such membranes of defined composition can be used to study lipid-protein interaction and to correlate membrane structure with composition. The number of particles observed by freeze-fracture electron microscopy can be correlated with protein content, whereas the percentage of smooth domain is proportional to the lipid content of the reconstituted membrane. Since 90% or more of the protein of the reconstituted membrane is the calcium pump protein, the number of particles observed by freeze-fracture is directly proportional to the amount of calcium pump protein in the membrane. The number of pump molecules calculated to be in the membrane is greater by a factor of two than the number of particles which we observed. This multiplicity ratio could be greater depending upon the assumptions made regarding the width of the membrane (see "Appendix"). Thus, it would appear that the particles consist of two or more molecules of pump protein. The change in protein concentration of the membrane is reflected also in thin sections and by negative staining. In thin sections, the broad inner and outer 70 A bands become discontinuous and patchy and, in the limit, approach a symmetrical 20,20,20 A trilayer as the protein content of the membrane becomes small. In an analogous fashion, the concentration of particles at the surface of the membrane, observed by negative staining, decreases with increasing lipid concentration in the membrane. Thus, the correlation of composition with structure can be observed by each of the three methods of sample preparation for electron microscopic analysis.


Albumin-like proteins from the pituitary glands of Dahl salt-resistant rats. Dahl selectively bred rats for susceptibility (S strain) or resistance (R strain) to the hypertensive effect of high salt (NaCl) diet. Pituitary glands of R rats accumulate large amounts of four unique proteins not seen in S rats. These proteins were called R1, R2, R3, and R4 in order of decreasing electrophoretic mobility. Albumin, R4, R2, and R1 all bound to an affinity column for albumin (Cibacron blue 3G-A dye coupled to agarose) and were eluted in that order by a KSCN gradient. It was shown by crossed immunoelectrophoresis that R1 and R2 cross-react with plasma albumin. Peptide maps or tryptic digest of R1 and albumin showed that the majority of peptides generated were identical. It was not possible to incorporate labeled amino acid into albumin or the albumin-like R proteins with pituitary incubates, indicating that albumin-like proteins were not synthesized de novo by pituitary glands. R rat pituitary glands showed much greater protease (arginine esterase) activity than did S. This suggests that R proteins are formed locally in the pituitary gland of R proteins are formed locally in the pituitary gland of R rats by cleavabe of specific peptide bonds in albumin. The function of these endogenous albumin fragments is unknown, but albumin fragments produced in vitro by other investigators are known to potentiate bradykinin, a hypotensive peptide.


Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase. We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.


The topological orientation of N,N'-diacetylchitobiosylpyrophosphoryldolichol in artificial and natural membranes. Purified N,N'-diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) in the presence of detergent was shown to act as a substrate for the soluble enzyme galactosyltransferase. The nature of this transfer reaction and the galactose-containing trisaccharide-lipid product have been partially characterized. Using galactosyltransferase as a probe, the topolical arrangement of chitobiosyl-lipid in both artificial and natural membranes has been examined. When incorporated into unilamellar liposomes made from phosphatidylcholine, the disaccharide residue of chitogiosyl-lipid adopts a random transbilayer orientation. Furthermore, no significant mobility in the transverse plane of the membrane (i.e. flip-flop) is detectable. Using both sealed total microsomes or isolated rough microsomes from hen oviduct, the topology of chitobiosyl-lipid after its biosynthesis in the membrane has been determined. The results suggest that, once formed, chitobiosyl-lipid is a relatively static component of the membrane and is oriented with its disaccharide residue facing the lumen of the endoplasmic reticulum.


Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis. Physical-chemical studies of pyruvate carboxylase from Pseudomonas citronellolis demonstrate that the enzyme has an alpha 4 beta 4 structure. The individual polypeptides, alpha (Mr = 65,000) and beta (Mr = 54,000), were separated and isolated by preparative gel electrophoresis. Analysis of the relationship between Coomassie blue staining and protein quantity for each polypeptide indicated that the alpha and beta subunits are present in a 1:1 stoichiometry in the native enzyme. Determinations of the molecular weight of the protein by sedimentation equilibrium (Mr = 454,000), gel filtration analysis (Mr = 510,000), disc gel electrophoresis (Mr = 530,000), and mass measurement from the Scanning Transmission Electron Microscope (Mr = 530,000) are consistent with the proposed alpha 4 beta 4 structure. Disc gel electrophoresis studies revealed that under certain circumstances the enzyme may dissociate to a smaller molecular weight species (Mr = 228,000). This dissociation phenomenon could explain the earlier reported observation of Taylor et al. ((1972) J. Biol. Chem 22, 7388-8390) that the enzyme had a molecular weight of 265,000. Evidence from electron microscopic studies shows that the three-dimensional structure of this enzyme is quite distinct from other species of pyruvate carboxylase. The enzyme does not show the typical rhombic appearance which has been noted for chicken liver, sheep liver, and yeast pyruvate carboxylase.