bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake mid Metabolism of Histidine during Stress
Authors: Huszti Zsuzsanna , Sourkes Theodore L.
Published: December 2009

The rate of formation of expired 14CO2 from histidine, 14C-labeled in the carboxyl group (C-His) or in the imidazole ring (R-His), and also from 14C-ring labeled urocanic acid (R-Uro) has been measured in rats subjected to restraint and cold stress. The rate of expiration of 14CO2 by restrained rats given C-His was significantly increased over control values; that from R-His was decreased. When restrained rats were also exposed to cold (4 °C), the rate of formation of 14CO2 from C-His decreased; that from R-His now showed an even greater decrease than when the measurements were made at room temperature. The rate of formation of labeled carbon dioxide from R-Uro was not altered significantly by these stressful procedures.Parallel measurements of the enzyme activities of histidine catabolism in rats restrained at room temperature for 2 h revealed no alteration in histidase activity of liver and lungs, but there was a marked increase in the histidine decarboxylase activity of stomach. Exposure of rats to cold over 1 h during the restraint of movement also resulted in enhanced histidine decarboxylase activity but of a lesser degree; 2 h of cold exposure during restraint abolished the difference between the control and stressed animals that had been observed at room temperature.The serum histidine concentration was measured 30 min after the intraperitoneal administration of 500 mg of histidine per kilogram body weight to rats. Both groups of stressed animals (restraint at room temperature, restraint in the cold) had much higher levels than observed in the corresponding controls. These results suggest that the increased histidine decarboxylase activity of stomach that develops under the stress of restraint is associated with a compensatory decrease in the rate of histidine uptake from the blood into the tissues. The data obtained for the rate of formation of expired 14CO2 have been interpreted in this light.
Evidence for an Asymmetric Distribution of Phospholipids in the Human Erythrocyte Membrane
Authors: Kahlenberg Arthur , Walker Caroline , Rohrlick Ruth
Published: December 2009

The changes in phospholipid composition of the inner (cytoplasmic) surface of the human erythrocyte membrane resulting from the digestion of sealed inside-out vesicles with phospholipases A2 and C were determined. Virtually all of the phosphatidylethanolamine and phosphatidylserine and 30–40% of the phosphatidylcholine and sphingomyelin of inside-out vesicles were found to be accessible to enzyme hydrolysis. In contrast, all of the above phospholipids of unsealed ghosts were susceptible to phospholipolytic digestion. These results are a direct demonstration of an asymmetric distribution of phospholipids in the human erythrocyte membrane.
Molecular Species of Ethanolamine Phosphatides of Dog and Pig Kidney
Authors: Yeung S. K. F. , Kuksis A.
Published: December 2009

The total ethanolamine phosphatides of the dog and pig kidney were isolated by T.L.C. (thin-layer chromatography) and the fatty acid methyl esters and dimethylacetals derived by transmethylation were identified by combined gas–liquid chromatography and mass spectrometry methods. It was shown that 40–50% of the ethanolamine phosphatides from both sources were piasmalogens in which the long-chain vinyl ethers were paired largely with arachidonic acid. Although the vinyl ethers of the pig kidney yielded mainly the palmitaldehyde, those of the dog kidney generated nearly equal proportions of palmit-, stear-, and oleoaldehydes. No evidence was obtained for the occurrence of nitrogenous bases other than ethanolamine in these phosphatide fractions. The significance of the above observations in relation to the potential anti-renin activity of the kidney phospholipids is pointed out.
Transport of Succinate in Escherichia coli. III. Biochemical and Genetic Studies of the Mechanism of Transport in Membrane Vesicles
Authors: Lo Theodore C. Y. , Rayman M. Khalil , Sanwal B. D.
Published: December 2009

The D-lactate oxidation dependent transport of succinate in membrane vesicles of an Escherichia coli strain lacking succinate dehydrogenase and fumarate reductase is inhibited by several categories of compounds. One category consists of compounds that are electron transport inhibitors (Amytal, Dicumarol, and mercurials), the second of compounds that act as competitive inhibitors of D-lactate dehydrogenase (oxamate and β-chlorolactate), the third of reagents that inhibit the Ca2+–Mg2+-activated ATPase (dicyclohexylcarbodiimide and pyrophosphate), and the fourth of compounds that tap off electrons from the respiratory chain (2,6-dichlorophenolindophenol). None of the succinate transport inhibitors, including mercurials like p-chloromercuribenzoate, interfere with the binding of succinate to the presumed membrane carriers.Membrane preparations from mutants of E. coli lacking D-lactate dehydrogenase are unable to transport succinate in the presence of D-lactate. Whole cells of these mutants, however, take up succinate normally. This observation suggests that D-lactate oxidation is not obligatorily linked in vivo to the uptake of succinate although the possibility is not excluded that transport in such mutants may be linked to some other dehydrogenase. Mutants having altered levels of ATPase, or membrane preparations made from such cells also have greatly reduced capacity to transport succinate. This observation coupled with the finding that ATPase inhibitors block dicarboxylate transport suggests involvement of ATPase in an unknown way in the concentrative uptake of succinate.With the exception of oxamate, β-chlorolactate (competitive inhibitors of D-lactate oxidation), and dicyclohexylcarbodiimide, all of the inhibitors of succinate uptake (including p-chloromercuribenzoate) cause an immediate efflux of preloaded succinate from membrane vesicles. Efflux is also caused by proton conducting reagents. The Km for efflux is 1.9 mM. This value is to be compared with the Km for influx, which is only about 0.02 mM.The weight of evidence favors the view that the active transport of succinate in vesicles occurs as a result of an energization of the membranes by the passage of electrons, although alternate oxidation and reduction of the succinate carrier as a mechanism for transport has not been definitely ruled out.
Biosynthesis of Spin-Labelled Lipids and Their Use in the Study of Biological Membranes: Isolation and Characterization of Membrane-Bound Spin-Labelled sn-3-Phosphatidic Acid-2-3H Prepared by Enzymatic Incorporation of Spin-Labelled Stearic Acid into sn-Glycero-3-phosphate-2-3H
Authors: Stanacev N. Z. , Stuhne-Sekalec L. , Schreier-Muccillo S. , Smith I. C. P.
Published: December 2009

Spin-labelled 12-doxylstearic acid was covalently incorporated into sn-glycero-3-phosphate-2-3H yielding sn-3-phosphatidic acid-2-3H, a key intermediate in phospholipid biosynthesis, in a reaction catalyzed by isolated guinea-pig liver microsomes. It was found that at least 17.1% of the obtained sn-3-phosphatidic acid-2-3H contained spin label. Degradation of spin-labelled sn-3-phosphatidic acid-2-3H with the phospholipase A from Crotalus adamanteus yielded both lyso-sn-3-phosphatidic acid-2-3H and free fatty acids which were paramagnetic, establishing that the incorporated 12-doxylstearic acid occupies both sn-1- and sn-2-positions of the biosynthesized sn-3-phosphatidic acid-2-3H.A procedure for the preparation and isolation of biosynthetically spin-labelled radioactive sn-3-phosphatidic acid bound to the microsomal membrane was developed and the concentrations of spin-label and tritium were quantitatively determined. This procedure is believed to have a general application in the study of biological membranes. The electron spin resonance spectra and their quantitation are given and discussed for the spin-labelled sn-3-phosphatidic acid-2-3H in solution and in the membrane-bound form. Some general requirements for covalent spin labelling of biological membranes are discussed.
Chain Elongation of Aromatic Amino Acids: the Role of 2-Benzylmalic Acid in the Biosynthesis of a C6C4 Amino Acid and a C6C3 Mustard Oil Glucoside
Authors: Dörnemann D. , Löffelhardt W. , Kindl H.
Published: December 2009

A chemical synthesis of specifically 14C-labelled 2-benzylmalic acid, hitherto unknown, was developed. 4-Phenylacetoacetate obtained by condensation of phenylacetyl chloride-1-14C with ethyl acetoacetate yielded 2-benzylmalic acid-2-14C after cyanohydrin reaction and hydrolysis.2-Benzylmalic acid-2-14C, administered to shoots of Nasturtium officinale and Barbarea vulgaris, was shown to be an efficient precursor of the aglucone moiety of the mustard oil glucoside gluconasturtiin. The incorporation of radioactivity agreed well with the values reported for incorporation of 3-benzylmalic acid, but was considerably higher than that obtained after application of L-phenylalanine-U-14C. A conversion of 2-benzylmalic acid into 3-benzylmalic acid and 2-amino-4-phenylbutyric acid could also be demonstrated. These findings provide the final evidence for a chain-lengthening mechanism leading to homologous amino acids as proposed by Underbill and Wetter in 1966.
Evidence for the Biosynthetic Difference between Isolated Mitochondria and Microsomes from Guinea-Pig and Rat Liver Regarding Lysophospharidic Acid, Phosphatidic Acid, CDP-Diglyceride, Phosphatidylglycerol, and Cardiolipin
Authors: Davidson J. B. , Stanacev N. Z.
Published: December 2009

The enzymatic activities of marker enzymes (NADPH – cytochrome c reductase and glucose-6-phosphatase) and synthetic enzymes (acyl-CoA:sn-glycero-3-phosphate acyltransferase, CTP:sn-3-phosphatidic acid cytidyltransferase, and CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase) were measured in both isolated mitochondria and microsomes from liver of guinea pig and rat. Results thus obtained show a significant difference in activities of these enzymes between subcellular particles within species and between two examined species. The activity of acyl-CoA:glycero-3-phosphate acyltransferase in guinea-pig mitochondria parallels the activity of microsomal marker enzymes in this fraction, while in rat liver mitochondria the activity is relatively higher and cannot be accounted for by the microsomal content as determined by marker enzymes. Implications of these results regarding mitochondrial autonomy in the biosynthesis of polyglycero-phosphatides and their precursors are discussed.
The Identification of Proinsulin-Synthesizing Polysomes in Fetal Bovine Pancreas
Authors: Hew Choy-L. , Yip C. C.
Published: December 2009

Antiserum against bovine pancreatic RNase A was produced in the rabbit. (Fab′)2 fragments were prepared from the antiserum and were found to inhibit the degradation of rat liver polysomes by fetal bovine pancreatic supernatant which contained RNase activity. Using this anti-RNase preparation, we were able to obtain fetal bovine pancreatic polysomes which appeared intact. Radioimmunoassay of the polysomal fractions after sucrose density gradient centrifugation showed that insulin immunoreactivity was associated mainly with tetrasomes, pentasomes, and some larger polysomal aggregates, suggesting the participation of these polysomes in the synthesis of proinsulin. The possibility that a precursor larger than proinsulin might be involved in the synthesis of insulin was discussed.
Occurrence of c-Type Cytochrome in Mycobacterium lepraemurium
Authors: Ishaque M. , Kato L.
Published: December 2009

The existence of c-type cytochrome in Mycobacterium lepraemurium was examined. The dithionite-treated cell-free extracts exhibited absorption peaks of cytochromes a + a3 and b, whereas the alpha band of c-type cytochrome at 552 nm was obscured by the large absorption peak of cytochrome b at 560 nm. The addition of NADH, NADPH, or succinate to cell-free extracts caused the reduction of b- and c-type cytochromes to nearly the same extent and thus the difference spectra displayed distinct separate peaks of b- and c-type cytochromes at 562 and 552 nm, respectively. The cell-free extracts treated with ascorbate showed absorption bands of cytochrome types c and a + a3, whereas the addition of succinate to a system preinhibited by antimycin A revealed the absorption bands of cytochrome b only. The absorption spectrum of the pyridine hemochromogens of M. lepraemurium was similar to that of mammalian cytochrome c. The results clearly indicated that, in addition to cytochromes of the a + a3 and b type, c-type cytochrome is also present in M. lepraemurium.
A Comparative Study of the Relative Ease of Thermolytic Depurination vs. Depyrimidination in 2′-Deoxynucleosides
Authors: Olafsson P. G. , Bryan A. M. , Davis G. C. , Anderson S.
Published: December 2009

Following fusion, 2′-deoxynucleosides undergo thermolytic cleavage to produce furfuryl alcohol and water along with the corresponding base. Contrary to previous suggestions, pyrimidine as well as purine deoxynucleosides are subject to base cleavage, the relative ease of thermolysis being 2′-deoxyguanosine > 2′-deoxyadenosine > 2′-deoxycytidine > 2′-deoxyuridine > thymidine. When dissolved in dimethyl sulfoxide, these nucleosides are susceptible to a similar cleavage at a lower temperature, with guanine being liberated from deoxyguanosine much more readily than adenine from deoxyadenosine, a feature which was in agreement with observations of Greer and Zamenhof (J. Mol. Biol. 4, 123 (1962)) regarding thermal depurination of DNA.
Electrophoretic Studies of the Proteins of Rat Liver Endoplasmic Reticulum
Authors: Bailey D. J. , Murray R. K. , Rolleston F. S.
Published: December 2009

The proteins of rough and smooth endoplasmic reticulum of rat liver were examined by electrophoresis in (1) a starch – urea – aluminum lactate system, which resolved 10 components; (2) a starch – urea – aluminum lactate – mercaptoethanol system which resolved 18 components; and (3) a polyacrylamide – sodium dodecyl sulfate (SDS) – urea – mercaptoethanol system, which resolved 33–34 components. In each system, the protein patterns of the membranes of the rough and smooth endoplasmic reticulum were found to be very similar. However, using polyacrylamide–SDS the rough endoplasmic reticulum was found to contain a protein of molecular weight 36 000, which was barely detectable in the smooth endoplasmic reticulum. Evidence is presented which suggests that this was a membrane component, although the possibility that it was a ribosomal protein could not be absolutely excluded. The membranes of the endoplasmic reticulum of male rats were also found to contain a protein of molecular weight 35 000, which was barely detectable in membranes from female rats.
Thermophilic Alanine Dehydrogenase from Halobacterium salinarium
Authors: Keradjopoulos D. , Wulff K.
Published: December 2009

Alanine dehydrogenase (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) from Halobacterium salinarium requires high concentrations of NaCl for both activity and stability. The enzyme is thermophilic with an optimum temperature of 70 °C in 3.4 M KCl and of 60 °C in 3.4 M NaCl. A thermophilic character has also been found for six other enzymes from H. salinarium. The alanine dehydrogenase becomes inactivated if the NaCl concentration drops below 1.5 M and it becomes reactivated if the salt concentration is increased to 4 M. Both reactions, the inactivation as well as the reactivation, require the presence of 2-mercaptoethanol for a complete reactivation. The reactivation reaction is irreversibly inhibited by N-ethylmaleimide.
Chemical Modifications of Escherichia coli Ribosomal Proteins S4, S7, and S8 in Regard to Their Capacity for Binding 16 S RNA
Authors: Lemieux Gérald
Published: December 2009

Mild specific chemical modifications were used to test the possible role of cysteines, tyrosines, and amino groups of ribosomal proteins S4, S7, and S8 for binding to 16 S RNA (Escherichia coli). The single cysteine residue present in proteins S4 and S8 is not directly involved in the binding process with RNA. The protein S7 does not contain cysteine. Total nitration of tyrosines in S4, S7, and S8 abolishes the binding capacity of these proteins. However, when the modification is done with the preformed protein–RNA complexes, some protection occurs. Two amino groups could be reacted in free S8 as well as in an S8–RNA complex. This modification does not influence protein–RNA interaction. Proteins S4, S7, and S8 in Regard to Their Capacity for Binding 16 S RNA. Can. J. Biochem. 52, modified. A new method for the stoichiometric determination of single protein–RNA complexes is presented.
Isolation of Somatostatin (a Somatotropin Release Inhibiting Factor) of Ovine Hypothalamic Origin
Authors: Brazeau P. , Vale W. , Burgus R. , Guillemin R.
Published: December 2009

Isolation of somatostatin, a tetradecapeptide of ovine origin inhibiting somatotropin secretion, is reported. About 490 000 hypothalamic fragments were submitted to alcohol–chloroform extraction, countercurrent distribution, ion-exchange chromatography, gel filtration, and partition chromatography. Of the 8.5 mg material thus obtained, 77% was accounted for by a peptide shown homogeneous by electrophoresis, thin-layer chromatography, and amino acid analysis. The peptide inhibits the secretion of radioimmunoassayable growth hormone at doses of ≥ 1.0 nM in vitro and 400 ng per rate in vivo.
The Hyperglycemic Effect of 1-Deoxy-D-manno-heptulose. Inhibition of Hexokinase, Glucokinase, and Insulin Release in Vitro
Authors: Leshem B. , Sharoni Y. , Dimant E.
Published: December 2009

Injection of 1-deoxy-D-manno-heptulose (1-DMH, 400 mg, subcutaneously) into rats is associated with a hyperglycemia which reaches a peak after 2 h and is about half that caused by the same dose of D-manno-heptulose (MH). Insulin release from pieces of rabbit pancreas suspended in 15 mM glucose and 20 mM 1-DMH was about 20% of the release in the absence of 1-DMH. Under the same conditions MH caused a complete block of insulin release. Yeast hexokinase was inhibited to the extent of 58% and 68% by 1-DMH and MH (14.3 mM), respectively. 1-DMH is also inferior to MH as an inhibitor of crude rat liver hexokinase and glucokinase. The primary hydroxyl group on carbon-1 of MH is not required for the heptulose to exert its inhibitory effect on hexokinase.
Glucagon Biosynthesis in Avian Pancreatic Islets: Evidence for Medium-Sized Biosynthetic Intermediates
Authors: Tung A. K.
Published: December 2009

Isolated pigeon pancreatic islets have been found to incorporate tritiated tryptophan into glucagon-immunoreactive components having molecular weights in the 6000–9000 range. It is suggested that these components may represent minor biosynthetic forms involved in the biosynthesis of glucagon in the avian islets.
Wheat-Embryo Ribonucleates. III. Modified Nucleotide Constituents in Each of the 5.8S, 18S and 26S Ribonucleates
Authors: Lau R. Y. , Kennedy T. D. , Lane B. G.
Published: December 2009

Modified nucleotide constituents in the NaCl-insoluble (2.5 M, 0 °C) fraction of wheat-embryo RNA (iRNA) have been efficiently labelled by imbibing wheat embryos in media that contain [32P]phosphate and/or [methyl-14C]methionine. Chromatographic analyses have shown that pseudouridylate and sugar-methylated nucleotides (each) account for a similar proportion (1–2 mol %) of the total nucleotides in alkali hydrolysates of each of the 5.8S, 18S and 26S components of wheat-embryo iRNA. All of the 16 possible sugar-methylated dinucleotide sequences (Nm-N) that can be formed by permuting the arrangement of the four classical ribonucleosides (N) and their O2′-methylated derivatives (Nm) are present in the 18S component, and all except Cm-G are present in the 26S component of wheat-embryo iRNA. Two sugar-methylated dinucleotide sequences (Am-A and Gm-C) are present in the 5.8S component. All of the (approximately seven) sugar-methylated trinucleotide sequences (Nm-Nm-N) are confined to the 26S component. The base-methylated nucleotide N6-monomethyladenylate is present in both 18S and 26S components, but the base-methylated nucleotide N6-dimethyladenylate is confined to the 18S component. An allied investigation of the NaCl-soluble (2.5 M, 0 °C) fraction of wheat-embryo RNA (sRNA) has shown that an alkali hydrolysate of the 5S component of the s[32P]RNA is virtually devoid of modified compounds.
Metabolism, Distribution, and Disappearance of Infected β-Phenylethylamine in the Rat
Authors: Wu P. H. , Boulton Alan A.
Published: December 2009

In the absence of a monoamine oxidase inhibitor, the bulk of intravenously injected radioactively labelled β-phenylethylamine was oxidized to phenylacetic acid. In the presence of pargyline, most of the label in tissues remained as unchanged phenylethylamine; small amounts of labelled phenylethanolamine, tyramine and octopamine were also identified. After intravenous injection of [14C]phenylalanine, only very small amounts of [14C]phenylethylamine could be located in urine and faeces. β-Phenylethylamine became concentrated in all tissues, including brain, following intravenous introduction both in the presence and absence of pargyline. Its clearance from these tissues and from brain regions was very fast.
Changes in Metabolism of Ethanolamine and Its Derivatives in Liver During Fasting
Authors: Haines D. S. M. , Derksen D. H.
Published: December 2009

The time course of incorporation of radioactivity into liver phosphatidylethanolamine and its precursors was studied following intraportal injection of [1,2-14C]ethanolamine into rats that had been fasted for 24 h and into nonfasted rats. Marked diminution in the labelling of liver phosphatidylethanolamine and CDP-ethanolamine was present in the fasted groups while the radioactivity contained in the ethanolamine and phosphoryl-ethanolamine pools of these livers was increased. The livers of the fasted rats contained significantly higher levels of ethanolamine, phosphorylethanolamine and CDP-ethanolamine while choline and phosphorylcholine were decreased. These changes are identical to those which we have described earlier in the livers of nonfasted rats fed a choline deficient diet.In a further experiment, the duration of the fasting period necessary to bring about these changes was studied. Incorporation of radioactivity into liver phosphatidylethanolamine at 2.5 min following intraportal injection of [1,2-14]ethanolamine declined progressively up to 7.5 h of fasting and did not decrease further after that time. There was a concomitant decrease in labelling of CDP-ethanolamine with retention of radioactivity in ethanolamine and phosphorylethanolamine. These results show that the liver metabolism of ethanolamine is altered quite early in the fasting period and suggest that it may be related in some way to the metabolic response to fasting.
RNA Polymerase Stimulation: Effect of Aldosterone and Other Adrenocorticoids on RNA Turnover in Rat Kidney
Authors: Mishra Ram K. , Feltham L. A. W.
Published: December 2009

The results of steroid hormone stimulation of aggregate RNA polymerase activity in kidney can be interpreted in terms of either template or enzyme alterations. In order to discover the effect of aldosterone on enzyme or DNA template, the RNA polymerases were purified from kidney nuclei of normal, adrenalectomized or adrenalectomized plus aldosterone treated rats and the activity was determined using different sources of DNA. It was found that the DNA from aldosterone treated rats was transcribed more efficiently than from other sources. Aldosterone does not seem to have a direct effect on the RNA polymerase. Fractionation of ([14C]aldosterone injected) kidney chromatin revealed the presence of radioactivity in the non-histone acidic proteins and DNA, suggesting the possible binding of aldosterone or aldosterone–receptor complex to these chromatin fractions which may result in enhanced template activity.Turnover of RNA was also studied in various subcellular fractions: nuclei, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, polysomes, ribosomes and sRNA, in normal, adrenalectomized, and adrenalectomized plus adrenocortical hormone treated rat kidney, by following the loss of radioactivity after a single injection of [14C]-orotic acid. Daily administration of aldosterone or deoxycorticosterone reversed the effects of adrenalectomy. Daily administration of corticosterone was without effect.
Degradation of RNA in Nuclei From Ehrlich Ascites Cells
Authors: Speers E. Ann , Tigerstrom Richard G. von
Published: December 2009

Nuclei were prepared from Ehrlich ascites cells in 80% yield by homogenization of the cells in an aqueous solution containing Triton N-101 and washing of the nuclear fraction by centrifugation and resuspension. Compared to the enzyme activities present in cell extracts, approximately 47% exo-RNase I, 15% alkaline RNase II, 9% acid RNase II and 7% acid phosphatase were associated with the nuclear fraction after isolation. Exo-RNase I and alkaline RNase II were rapidly lost from nuclei during incubation at 37 °C. The degradation of newly synthesized RNA in nuclei incubated at 37 °C was followed by polyacrylamide gel electrophoresis and by characterization of acid-soluble degradation products. The rate of hydrolysis of the nuclear RNA was rapid during the initial stages of incubation and then proceeded at a much reduced rate. Nucleoside 5′-phosphates were the major acid-soluble degradation products, in agreement with the presence of exo-RNase I. Although a considerable amount of alkaline RNase II was associated with the nuclear fraction, extensive endonucleolytic cleavage of the nuclear RNA was not apparent. Compared to the processing of nuclear RNA in whole cells, however, the degradation in isolated nuclei was relatively non-specific.
A Simple Chromatographic Method for the Determination of the Methylated Basic Amino Acids in Proteins
Authors: Zarkadas Constantinos G.
Published: December 2009

A chromatographic method is described for the determination of ε-N-monomethyl-, ε-N, ε-N-dimethyl-, and ε-N, ε-N, ε-N-trimethyllysines, 1-methyl-, and 3-methylhistidines, NG,NG-dimethyl-, and NG,N′G-dimethylarginines, ornithine, the diastereoisomers of hydroxylysine, basic amino acids and related compounds, using Durrum type DC-6A 11.0 ± 1.0 μm spherical resin. The complete separation of all of these amino acids as discrete peaks in the Beckman 120B amino acid analyzer was achieved for the first time using a single column (60 × 0.9 cm), one buffer system (0.35 N sodium citrate) adjusted at either pH 5.734 or 5.657 ± 0.002, one buffer flow rate (30 ml/h), and one temperature (28 °C). This procedure was used for the analysis of these unusual amino acids in protein hydrolysates of histones and collagen and can be applied to the determination of these compounds in hydrolysates of plant and animal tissues.
Effect of Triphenylmethane Derivatives on Cell-Free Maeromolecular Synthesis. I Aminoacyl-tRNA Synthetase
Authors: Igarashi S. J. , Zmean J. A.
Published: December 2009

The inhibitory effect of aurintricarboxylic acid (ATA) on phenylalanyl-tRNA synthetase is demonstrated in the rabbit-reticulocyte system. This inhibition is not specific to ATA; other triphenylmethane derivatives are also potent inhibitors of this enzyme reaction. The site of inhibitory action is in the enzyme itself, not in the tRNA molecule. It thus appears that the skeletal structure of ATA, not the side chains, is responsible for its inhibitory action, and that ATA is a nonspecific inhibitor of the reactions involving polynucleotides.
Studies on Molecular Weights of Two Peptide Hormones from the Urophysis of White Sucker (Catostomus commersoni)
Authors: Moore Graham , Letter Anita , Tesanovic Maria , Lederis Karl
Published: December 2009

The molecular weights of two active principles extracted from the urophysis of the teleost fish Catostomus commersoni in 0.1 N HCl or in 0.25% acetic acid have been investigated by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis. Two peptides with urotensin I (long-acting rat hypotensive) activity and two peptides with urotensin II (fish smooth muscle stimulating) activity were found by these procedures. The smaller of the two urotensin I peptides (molecular weight 1200–1700), designated urotensin IS, was shown to be a fragment of the larger peptide (molecular weight 2300–3000) which is produced by acid hydrolysis without loss of rat hypotensive activity. The two urotensin II peptides are suggested to represent either a monomer and a dimer or open and closed forms of a peptide.
Coupling of Glycine and Alanine Transport to Respiration in Cells of Escherichia coli
Authors: Sprott G. D. , Dimock K. , Martin W. G. , Schneider Henry
Published: December 2009

Energy coupling for uptake of glycine and alanine in glycerol grown cells of Escherichia coli differs from that of the aromatic amino acids. Respiration and uptake of glycine and alanine show similar inactivations in cells exposed to high intensity violet light or to various concentrations of cyanide. In contrast, uptake of phenylalanine, tyrosine, and tryptophan is resistant to effects of light or cyanide. Anoxia largely inhibits uptake of glycine and alanine while that of the aromatic amino acids is only partially affected. Furthermore, ferricyanide (but not ferrocyanide) completely restores active uptake of aromatic amino acids under anoxic conditions but is without effect on glycine and alanine uptake. Adenosine 5′-triphosphate (ATP) concentration does not increase in anoxic cells exposed to ferricyanide, indicating that ATP cannot be responsible for this restoration. The data suggest that glycine and alanine represent amino acids whose transport shows a complete dependence on energy derived from respiration, while the energy for transport of the aromatic amino acids may be obtained from other sources.
The Reaction of Chymotrypsin with 2,3-Butanedione Trimer
Authors: Fliss H. , Tozer N. M. , Viswanatha T.
Published: December 2009

A method for the preparation of the trimer of 2,3-butanedione has been developed. The reaction of this trimer with chymotrypsin Aα was examined in the presence or absence of light. Under conditions of exclusion of light, modification of one to two arginine residues and of a similar number of lysine residues could be achieved without any loss of enzymatic activity. The trimer facilitated a rapid photoinactivation of the enzyme with little or no modification of the above amino acid residues. Such photoinactivation was not promoted by the monomer 2,3-butanedione. Enzyme irradiated in the presence of the trimer was found to react with proflavine and diisopropylfluorophosphate to an extent greater than that expected on the basis of residual activity present. Proflavine protected the enzyme from the trimer promoted photoinactivation.
Purification and Some Properties of the Soluble and Membrane-Bound Adenosine Deaminases of Micrococcus sodonensis ATCC 11880 and their Distribution within the Family Micrococcaceae
Authors: Pickard M. A.
Published: December 2009

In Micrococcus sodonensis and some other Micrococcus species, adenosine deaminase is present both as a membrane-bound and a soluble enzyme. The membrane-bound adenosine deaminase can be extracted with n-butanol, and may account for up to 5% of the total cellular adenosine deaminase activity. In a number of comparative tests, no differences between the two enzyme forms could be found, thus they are believed to be similar molecular species. The purified membrane-bound or soluble enzyme had a molecular weight, obtained by gel-filtration, of 130 000 and was inactive toward adenine and adenine mononucleotides. It appears, therefore, to be more closely related to the calf-intestine enzyme than the Aspergillus oryzae form in respect to size and substrate specificity. Attempts to correlate membrane-bound adenosine deaminase activity with adenosine transport in isolated membrane vesicles of M. sodonensis indicated no obvious relationship between the two activities.
Magnetic Resonance Studies of Concanavalin A: Conformational Changes Induced by Ca2+ and α-Methyl-D-mannopyranoside
Authors: Barber Brian H. , Carver Jeremy P.
Published: December 2009

Three independent solution spectroscopic techniques (solvent proton relaxation enhancement, circular dichroism, and high resolution 220 MHz proton magnetic resonance spectroscopy) have been utilized to demonstrate metal ion- and monosaccharide inhibitor-induced structural perturbations for the dimeric form of the plant lectin concanavalin A (Con A). The results indicate that (i) the occupation of the transition metal ion site S1 by Mn2+ or Zn2+ does not detectably perturb the demetallized protein conformation, (ii) the binding of Ca2+ to the Con A – Mn2+ or Con A – Zn2+ complexes perturbs the protein structure in the vicinity of the S1 site as well as at points remote from the S1–S2 double ion site, and (iii) the binding of the monosaccharide inhibitor α-methyl-D-mannopyranoside to the fully metallized Con A complex also significantly perturbs the structural features of the protein.A detailed radio frequency dependence analysis of the Ca2+ effect on the solvent proton relaxation enhancement properties of the Con A – Mn2+ complex indicates that the considerable reduction in the observed enhancement upon Ca2+ binding principally results from an approximate 120-fold decrease in the single Mn2+ water of hydration exchange rate. The 220 MHz proton magnetic resonance spectra for Con A indicate that this form of spectroscopy is the most useful of those utilized in detailing the solution structural features of this lectin, and a tentative assignment for the C-2-H proton of histidine residue 24 (the S1 site ligand) has been proposed.
Oligonucleotides Containing Phosphotriester Internucleotidic Groups as Novel Substrates for Polynucleotide Enzymes
Authors: Narang S. A. , Itakura K. , Katagiri N.
Published: December 2009

Synthetic oligonucleotides containing internal 3′,5′-phosphotriester groups have been found to act as substrates for the enzymes polynucleotide kinase induced by bacteriophage T4 and terminal deoxynucleotidyi transferase.
Biosynthesis of Gliotoxin and Mycelianamide
Authors: MacDonald J. C. , Slater G. P.
Published: December 2009

Mycelium of Penicillium terlikowski was permeable to both L-[carboxyl-14C]phenylalanine and cyclo L-[carbonyl-14C]phenylalanyl-L-seryl. The former compound was incorporated well and the latter poorly into gliotoxin produced by stationary cultures of the organism. Mycelium of Penicillium patulum was permeable to L-[carboxyl-14C]tyrosine and to the cyclic dipeptides cyclo L-alanyl-L-[carbonyl-14C]tyrosyl and cyclo L-alanyl-D-[carbonyl-14C]tyrosyl. These cyclic dipeptides were not incorporated into mycelianamide produced by stationary cultures of the organism, but both D- and L-[carboxyl-14C]tyrosine were incorporated. The latter was incorporated in a specific manner into part of the mycelianamide molecule. The results did not support the postulate that the appropriate cyclic dipeptides, as outlined above, were free intermediates in the biosynthesis of gliotoxin or mycelianamide.
Distribution of Dipeptidase Activity between Lysosomes and Soluble Fraction of Rat Liver
Authors: Taylor Steve L. , Tappel Al L.
Published: December 2009

Dipeptidase activity toward Arg-Phe, Arg-Gly, and Trp-Leu exhibited bimodal distribution in the lysosomal and soluble fractions of rat liver. The majority (50–70%) of the dipeptidase activity was present in the soluble fraction. Some evidence for a plasma membrane dipeptidase, which hydrolyzes Trp-Leu but not Arg-Phe or Arg-Gly, also was found. The lysosomal dipeptidase activity had a pH optimum of 6.0–7.0, and was activated by sulfhydryl reagents. Lysosomal localization for some of the dipeptidase activity was established with Triton WR-1339 fractionation and latency experiments.
Distribution of Newly Formed Palmitate and Stearate Among Molecular Species of Choline and Ethanolamine Phosphatides
Authors: Kuksis A. , Myher J. J. , Marai L. , Yeung S. K. F. , Steiman I. , Mookerjea S.
Published: December 2009

The choline and ethanolamine phosphatides derived from isolated rat livers during perfusion with 75% deuterated water (Kuksis, A., Myher, J. J., Marai, L., Yeung, S. K. F., Steiman, I. &Mookerjea, S. (1975) Can. J. Biochem. 53, 509–518) were resolved into molecular species by argentation thin-layer chromatography. The time course of percentage replacement of the newly synthesized fatty acids in each molecular species was determined by gas chromatography with mass spectrometry. The results confirmed the earlier postulated differential utilization of palmitic and stearic acids in glycerolipid biosynthesis as well as supported the hypothesis of a precursor–product relationship between the oligoenoic and tetraenoic species of both phosphatides. Calculations of half-lives gave values of 14–19 h for palmitoyl oligoenes, 40–50 h for palmitoyl tetraenes, and 22–28 h for palmitoyl hexaenes of both choline and ethanolamine phosphatides. The corresponding stearoyl species had half-lives which ranged from 89 to 200 h. Evidence was obtained for a metabolic heterogeneity among subsets of molecular species recognized on the basis of combinations of new and old glycerol and fatty acids in the same glycerolipid molecule.
Characterization of Malate Dehydrogenase Isozymes in Wheat Germ
Authors: Legris A. J. , Tsai C. S.
Published: December 2009

Malate dehydrogenase of wheat germ exists in multiple molecular forms (isozymes). Comparisons of some physical properties such as Stoke's radii, sedimentation constants, electrophoretic mobilities on polyacrylamide gel, chromatographic behaviors on DEAE-cellulose, stabilities to heat and iodoacetamide inactivation, as well as kinetic parameters were described. When all these properties are considered together, at least five isozymes were found to associate with cytoplasm, mitochondria, glyoxysomes and proplastids of wheat germ.Wheat germ malate dehydrogenases are specific for the reduction of oxaloacetate and its monoesters. At least one carboxylic group of oxaloacetate must be free, in order to exhibit substrate activity, and maximum binding of oxaloacetate is achieved when both carboxylic groups are free. Soluble malate dehydrogenase and organelle-associated malate dehydrogenase can be differentiated readily in that the former can not utilize 4-ethyl oxaloacetate as the substrate. The organelle associated MDHs can be further differentiated by the mode of ATP inhibition.
Carboxylesterases (EC 3.1.1). The Molecular Sizes of Chicken and Pig Liver Carboxylesterases
Authors: Inkerman Peter A. , Winzor Donald J. , Zerner Burt
Published: December 2009

The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200, we conclude that the monomeric molecular weight is ~65 000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer–trimer system were estimated to be 0.02 l2 g−2 at pH 4 (concentration-dependent molecular weight data) and 2 × 105 l2 g−2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight ~65 000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in β-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of ~65 000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than ~65 000 for the pig enzyme, but ca. 50% of the chicken esterase is dissociated into two species of molecular weight ~30 000.
Carboxylesterases (EC 3.1.1). A Comparison of Some Kinetic Properties of Horse, Sheep, Chicken, Pig, and Ox Liver Carboxylesterases
Authors: Stoops J. K. , Hamilton S. E. , Zerner Burt
Published: December 2009

A comparative study of the kinetic behavior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificities towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis–Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simple scheme which accounts for both activation and inhibition is discussed. pH–kcat profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pKa's of 4.75 and 5.0, respectively.
Relationship Between RNA Polymerase and Protein Kinase Activities in Rat Mammary Gland Nuclei
Authors: Desjardins P. R. , Mendelson I. M. , Anderson K. M.
Published: December 2009

1. Extracts from rat mammary gland nuclei contain cyclic AMP - independent protein kinases which phosphorylate casein rather than histone.2. A major increase in nuclear protein kinase activity occurred during late pregnancy and was maintained with the onset of lactation.3. Two major peaks of activity were resolved by chromatography of nuclear extracts on DEAE-Sephadex; the first (NI) appeared in the void volume and the second (NII) was eluted by 0.05–0.12 M ammonium sulfate. Several other regions of lesser activity were also present.4. Protein kinases in the cytosol 105 000 × g supernatant, precipitated by 70% ammonium sulfate, dialyzed against buffer, and chromatographed on DEAE-Sephadex, yielded a major peak in the void volume and a minor component at about 0.20 M ammonium sulfate. Both components phosphorylated histone in preference to casein, and this was stimulated by cyclic AMP if histone was the substrate, but only the first (void volume) fraction was cyclic AMP -dependent when casein was used.5. Most of RNA polymerases Ib and II, derived from the nucleolus and nucleoplasm, respectively, appeared in column fractions distinct from those containing the major NI and NII protein kinases.6. Cyclic AMP altered the amount of RNA product synthesized by polymerases Ib and II, but the explanation for this is unknown. Due to their elution profiles and cyclic AMP - independence, protein kinases NI and NII are excluded from playing a catalytic role in these effects; participation of quantitatively minor protein kinases which co-elute with polymerase Ib and II is not yet excluded.
A Radio-Gas Chromatographic Method for Determining the Specific Radioactivity of Glycolic Acid in 14C-Labeled Leaf Tissue
Authors: Mahon J. D. , Egle K. , Fock H.
Published: December 2009

A method for the extraction and quantitative determination of both the mass and radioactivity of glycolic acid from 14C-labeled leaf tissue is described. The recoveries of both mass and radioactivity from standard [1-14C]glycolic acid solutions averaged 98%, and recovery of radioactivity added to plant samples as [1-14C]glycolic acid was over 90% after the complete procedure. The method was reliable with total samples containing as little as 130 nmol of glycolic acid.The mass of glycolic acid recovered from sunflower leaf tissue was proportional to the amount of tissue extracted. In experiments with different plant material, the amount of glycolic acid varied between 530 and 1120 nmol/dm2 of leaf tissue. The specific radioactivity of the glycolic acid in sunflower leaf tissue during photosynthesis in 14CO2 was never more than 20% of the specific radioactivity of the 14CO2 supplied.
The Lipopolysaccharides of Neisseria gonorrhoeae Colony Types 1 and 4
Authors: Perry Malcolm B. , Daoust Virginia , Diena Benito B. , Ashton Fraser E. , Wallace Rebecca
Published: December 2009

The lipopolysaccharides (LPS) of strains of Neisseria gonorrhoeae grown in type 1 (T1) and 4 (T4) colony forms have been isolated. LPS from T4 colony type cells on mild hydrolysis gave a lipid A and a core oligosaccharide composed of 2-amino-2-deoxy-D-glucose, D-glucose, D-galactose, L-glycero-D-manno-heptose and 3-deoxy-D-manno-octulosonic acid that appeared to be common to all the strains examined. LPS from T1 colony type cells on mild hydrolysis gave a lipid A and high molecular weight O polysaccharides which showed considerable differences in glycose composition for each strain examined. In those strains examined, T4 cells appear to produce a common 'R' type LPS whereas T1 cells produce an 'S' type LPS with structurally different O polysaccharide structures which probably account for serologically differentiated strains of N. gonorrhoeae.
Glutamic–Pyruvic Transaminase Activity Related to Red Blood Cell Age
Authors: Hubert C. , Poon M.-C.
Published: December 2009

Red blood cell glutamic–pyruvic transaminase (GPT) phenotypes and catalytic activities were studied in normal subjects and in patients with various hemolytic syndromes associated with reticulocytosis. To assess the effect of cell age on GPT activity, young cells were separated from older cells by centrifugation, and the catalytic activities were compared. In normal blood, there was a progressive fall in GPT activity from the top layer (younger cells) to the bottom layer (older cells), with a mean ratio of 1.90 ± 0.42. Similarly, in the blood of patients with reticulocytosis, the enzyme activity of the reticulocyte-rich layer was higher than that of the layer containing older cells (mean ratio 1.94 ± 0.95).
Analysis of O2′-Methylnucleoside 5′-Phosphates in Snake Venom Hydrolysates of RNA: Identification of O2′-Methyl-5-Carboxymethyluridine as a Constituent of Yeast Transfer RNA
Authors: Gray M. W.
Published: December 2009

Snake venom phosphodiesterase liberates the O2′-methylnucleoside (Nm) constituents of RNA as the corresponding 5′-nucleotides (pNm), which, in contrast to normal 5′-nucleotides (pN), are resistant to dephosphorylation by venom 5′-nucleotidase. This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA. The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry. Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined. The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms.During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2′-methylnucleoside 5′-phosphates. Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2′-methyl-5-carboxymethyluridine (cm5Um). Venom hydrolysis of yeast tRNA liberates the 5′-nucleotide of cm5Um in the form of a carboxyl-blocked derivative (pU-2). During alkaline hydrolysis of yeast tRNA, the blocking group in U-2 is labilized and cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap. The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol%, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2′-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast.In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2′-methylribose in yeast tRNA (Gray, M. W. &Lane, B. G. (1967) Biochim. Biophys. Acta 134, 243–257).