bluuebunny/crossref_metadata_2025 huggingface dataset:
α-Aminobutyronitrile as an Intermediate in Cyanide Fixation by Rhizoctonia solani
Authors: Mundy B. P. , Liu F. H. S. , Strobel G. A.
Published: December 2009

α-Aminobutyronitrile-14C was isolated from cultures of Rhizoctonia solani that had been administered H14CN. A pathway for H14CN utilization by this fungus involving the condensation of ammonia, HCN, and propionaldehyde to α-aminobutyronitrile is proposed. Enzymatic evidence shows that α-aminobutyronitrile is hydrolyzed to α-aminobutyric acid.
UDP-Galactose: Glycoprotein Galactosyl Transferase and UDP-N-acetylgalactosamine: Protein N-acetylgalactosaminyl Transferase Activities of Human Cerebrospinal Fluid
Authors: Ko G. K. W. , Raghupathy E. , McKean C. M.
Published: December 2009

The properties of UDP-galactose: glycoprotein galactosyl transferase and UDP-N-acetylgalactosamine: protein N-acetylgalactosaminyl transferase of human cerebrospinal fluid (C.S.F.) have been studied. The C.S.F. enzymes resemble closely the galactosyl and N-acetylgalactosaminyl transferases present in rat brain microsomes in ionic requirements, in sulfhydryl nature, and in acceptor specificities. The apparent Km value for UDP-galactose is 6.1 μM, and that for UDP-N-acetylgalactosamine is 4.0 μM. The C.S.F. transferases are in soluble forms and there are no detectable endogenous acceptors in C.S.F. for the two glycosyl transferases.Phenyllactate, phenylacetate, phenylpyruvate, and O-hydroxyphenylacetate (2–10 mM) inhibit the activities of both C.S.F. glycosyl transferases whereas phenylalanine and tyrosine, at the same concentrations, have no discernible effects on the transfer reaction. Phenyllactate and phenlacetate at concentrations of 6 and 10 mM, inhibit the activity of galactosyl transferase in C.S.F. by about 50% and 90%, respectively, but are without significant effects on the soluble galactosyl transferases of human serum, milk, and colostrum. Similar results are obtained for N-acetylgalactosaminyl transferase.
Demonstration of 2-Oxoglutarate:Glyoxylate Carboligase Activity in Tetrahymena pyriformis
Authors: Zaror-Behrens Gloria , Mavrides C.
Published: December 2009

A 2-oxoglutarate:glyoxylate carboligase, requiring thiamine pyrophosphate and magnesium, was demonstrated in Tetrahymena pyriformis and appears to be located in the mitochondrial fraction. The product of the reaction α-hydroxy-β-ketoadipate decarboxylates in the presence of acid to δ-hydroxylevulinate. The latter was identified in the acidified reaction mixtures. The carboligase activity varies coordinately with 2-oxoglutarate dehydrogenase. The two activities may reside in the same complex as in beef-heart mitochondria.
Inhibition of Ribonuclease Activity by Bentonite
Authors: Jacoli G. G. , Ronald W. P. , Lavkulich L.
Published: December 2009

The inhibition of ribonuclease activity by bentonite and the adsorption of the protein molecule within the clay matrix were assessed by enzyme and X-ray diffraction analysis, respectively.Pretreatment of bentonite with EDTA, potassium, barium, and barium–EDTA caused varying expansion of the d(001) spacing of the clay. The d(001) variation was sensitive to pH.Inhibition of the enzyme activity generally followed the pattern of expansion of the d(001) spacing of the clay, but failed when the interlayers of bentonite expanded beyond their maximum capability.Vermiculite, which is a clay having a higher cation exchange capacity than bentonite, did not expand from the normal state after similar treatment nor did it inhibit ribonuclease activity.When the secondary and tertiary structures of the protein molecule were disrupted, the denatured protein still entered the interlayers of bentonite, but caused a greater expansion of the d(001) spacing than the native ribonuclease.
Some Properties of α-Chymotrypsin and β-Galactosidase Supported in Polyacrylamide Gels
Authors: Bunting Peter S. , Laidler Keith J.
Published: December 2009

A study has been made of the characteristics of α-chymotrypsin and β-galactosidase trapped in polyacrylamide gels. Two methods were used to produce the gels: γ-irradiation and chemical polymerization, ammonium persulfate being used as catalyst in the latter case. The enzyme activities passed through maxima as the acrylamide concentration was increased. The supported β-galactosidase produced by chemical polymerization was approximately as active as that produced by γ-irradiation; the γ-irradiated preparation was more active in the case of α-chymotrypsin. The supported enzymes were somewhat less stable than the free enzyme.
"Metabolic Memory": Effect of Antecedent Dietary Manipulations on Subsequent Diet-Induced Response of Rats. I. Effects on Body Weight, Food Intakes, Glueose-6-Phosphate Dehydrogenase, and Malic Enzyme
Authors: Szepesi B.
Published: December 2009

Starvation followed by refeeding a 65% glucose diet led to elevated levels of hepatic glucose-6-phosphate dehydrogenase and malic enzyme activity. A second starve–refeed episode led to even greater enzyme responses. Food intake and body weight gain were increased by one, and further increased by a second, starve–refeed episode. Prefeeding a 65% fructose or sucrose diet ad libitum (which results in elevated enzyme levels comparable with the effect of one starve–refeed episode) was not accompanied by increased food intake or body-weight gain, nor did such treatment increase the enzyme response to subsequent starvation–refeeding. Previous starvation, however, did increase the subsequent enzyme response to feeding ad libitum a diet containing sucrose or invert sugar. The enzyme response was greater when the sucrose-containing diet was fed, and the diet effect was independent of the treatment effect. The results suggest that: (1) the metabolic consequences of starvation–refeeding are longer lasting than the metabolic consequences of feeding fructose or sucrose, (2) the effect of dietary sucrose is greater than the effect of an equal amount of invert sugar, (3) the metabolic effects of dietary fructose and sucrose may be mediated by different mechanisms than the corresponding effects of starvation–refeeding.
γ-Glutamyl Transpeptidase of Rat Kidney. Some Properties and Kinetic Constants
Authors: Elce John S. , Bryson Joan , McGirr L. G.
Published: December 2009

γ-Glutamyl transpeptidase has been partially purified from kidneys of adult male rats by solubilization with deoxycholate and chromatography in the presence of Triton X-100. A new, sensitive assay has been developed to study the reaction of glutathione with methionine in the micromolar range. This assay was found to differ from those using γ-glutamylnaphthylamide as substrate in the activation of the enzyme by anions and cations, and in terms of the nature of the reaction catalyzed by the enzyme. The kinetics of the transpeptidation reaction have been studied with the substrates glutathione and methionine. The pattern of inhibition caused by the product cysteinylglycine suggested that the reaction was non-sequential. The Michaelis constants for glutathione and methionine were of the order of 40 μM and 3 mM, respectively.
Amino Acid Analyses of Chymotrypsin-Like Proteases From the Sea Anemone (Metridium senile)
Authors: Stevenson Kenneth J. , Gibson David , Dixon Gordon H.
Published: December 2009

Amino acid analyses of two chymotrypsin-like serine proteases (designated proteases A and B) isolated from the gastric tissue of the sea anemone (Metridium senile) were conducted. Proteases A and B are distinct enzymes and are not related by minor proteolytic digestion. These enzymes contain similar total amino acid compositions of about 240 residues. The findings in protease A of a very high proline content (30 residues) and six residues of an unique amino acid, 2-aminoethylphosphonic acid (2-AEP; H2NCH2CH2PO3H), are of interest. 2-AEP was characterized on the amino acid analyzer and on high-voltage ionophoresis at pH 6.5. In protease B, four residues of 2-AEP were present and an anomaly was observed with the arginine residues.
Synthesis of Peptides by the Solid Phase Method. I. Tetradecapeptide Renin Substrate and Intermediate Peptides, Nonapeptides, and Fragments of Angiotensin II
Authors: Park W. K. , Choi C. , Rioux F. , Regoli D.
Published: December 2009

Tetradecapeptide renin substrate (H∙Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser∙OH) (1–14), tridecapeptide (1–13), dodecapeptide (1–12), undecapeptide (1–11), two nonapeptides (1–9) and (1–9-Leu), heptapeptide (1–7), tetrapeptide (1–4), tetrapeptide (5–8), pentapeptide (4–8), hexapeptide (3–8), and heptapeptide (2–8) were synthesized by the solid phase method and by using an improved reaction vessel. The yield averaged 50–70%. Homogeneity and purity of peptides were established with elemental anlysis for C, H, and N, paper chromatography in three solvent systems, electrophoresis, amino acid analysis, and enzymatic degradation by aminopeptidase M.
Synthesis of Peptides with the Solid Phase Method. II. Octapeptide Analogues of Angiotensin II
Authors: Park W. K. , Choi C. , Rioux F. , Regoli D.
Published: December 2009

Forty-six analogues of angiotensin II were obtained with the solid-phase method for peptide synthesis. The peptides were purified, using the conventional procedures; homogeneity and purity were established after paper, thin-layer chromatography, paper electrophoresis, amino acid analysis, elemental analysis, and enzymatic degradation by aminopeptidase. The biological activity of all compounds was compared with that of angiotensin II on the blood pressure of anesthetized rats. The same test was used to establish the antagonistic effect of several analogues against angiotensin II.
Purification and Properties of Acid Phosphatase I From the Cellular Slime Mold Polysphondylium pallidum
Authors: Horgen Ilona A. , Horgen Paul A. , O'Day Danton H.
Published: December 2009

A procedure for the purification of a phosphomonoesterase, designated as acid phosphatase I, from the cellular slime mold Polysphondylium pallidum is described. Ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography are utilized in this purification method. The enzyme was judged to be homogeneous by gel filtration and by acylamide gel electrophoresis. The molecular weight of the enzyme was estimated by gel filtration and density gradient centrifugation to be 150 000 daltons. Acid phosphatase I was shown to be relatively heat stable, and it lost no activity when kept at 4 °C, pH 7.35, for over 30 days. The pH optimum was 3.5, but the enzyme was found to be more stable when kept near neutral hydrogen ion concentrations. P. pallidum acid phosphatase I was most effective using the natural substrates, fructose-1,6-pbosphate, β-glycerolphosphate, and 5′-mononucleotides. Various compounds including known phosphatase inhibitors were tested as to their effect on the activity of the enzyme. The slime-mold acid phosphatase appears in many ways to be a typical acid phosphomonoesterase.
Characterisation of Nuclear and Cytoplasmic DNA Polymerases From Rat Intestinal Mucosa
Authors: Poulson Rozanne , Krasny J. , Zbarsky S. H.
Published: December 2009

Some properties of two DNA polymerases isolated from purified nuclei of rat intestinal mucosa were compared with those of a cytoplasmic DNA polymerase from the same tissue. The molecular weight of the NaCl-soluble nuclear polymerase was estimated to be 39 000, while a value of 100 000 was determined for both the Tris-soluble nuclear and cytoplasmic enzymes. The high molecular weight DNA polymerases shared many other common characteristics. Their chromatographic properties on DEAE-cellulose and phosphocellulose were identical. In the presence of 50 mM salt or 1 mM nalidixic acid the activities of the Tris-soluble nuclear and cytoplasmic enzymes were inhibited, whereas the activity of the low molecular weight NaCl-soluble nuclear enzyme was greatly enhanced. The activities of the three DNA polymerases were inhibited by sulfhydryl blocking agents. The NaCl-soluble nuclear enzyme showed maximal activity when native DNA that had been treated briefly with DNase I was supplied as the primer–template. Extensive treatment of the native DNA with DNase I, which converted 15% of it to acid-soluble form, reduced its templating activity by more than 90%. No incorporation was detected when denatured DNA was supplied as primer–template. In contrast, both native and denatured DNA served as primer–templates for the Tris-soluble nuclear and cytoplasmic DNA polymerases, and the most efficient primer–template tested was native DNA that had been rendered 15% acid soluble by digestion with DNase I.
Replicating DNA of Adenovirus Type 2
Authors: Bourgaux-Ramoisy Danielle , Robin Jean , Bourgaux Pierre
Published: December 2009

We have investigated the nature of the DNA synthesized in KB cells 24 h after infection with adenovirus type 2 (ad. 2). Cellular DNA synthesis being strongly depressed, a new molecular form of ad. 2 DNA could be detected in such cells in addition to mature viral DNA. This new form, referred to as DNA-R, is preferentially labelled after short pulses of 3H-thymidine, while the radioactive label can be chased into mature viral DNA. The sedimentation properties of DNA-R are those expected for linear replicating molecules having 1–2 times the molecular weight of mature viral DNA. As a result of being partly single-stranded, these molecules can be separated from mature viral DNA by chromatography on benzoylated-naphthoylated DEAE-cellulose. Purified DNA-R was examined under the electron microscope and found to consist of Y-shaped molecules. Thus DNA-R has the properties expected for linear monomeric molecules of ad. 2 DNA replicating undirectionally.
Mammalian Fatty Acid Synthetase. I. Purification and Properties of Human Liver Complex
Authors: Roncari Daniel A. K.
Published: December 2009

The human liver fatty acid synthetase complex was obtained in highly purified form as judged by agarose gel filtration chromatography, analytical ultracentrifugation, and polyacrylamide disc gel electrophoresis. On the basis of sedimentation equilibrium, its molecular weight was computed to be 410 000 ± 20 000. The major product was palmitic acid. One mole of the prosthetic group, 4′-phosphopantetheine, was present per mole of synthetase.Both acetyl-CoA and butyryl-CoA were effective primers of fatty acid synthesis. Under appropriate conditions, the intermediates, acetyl – and malonyl – fatty acid synthetase could be isolated. On the basis of pretreatment of the synthetase with N-ethylmaleimide and exposure of the intermediates to performic acid, it was determined that the complex had both thiol and non-thiol acyl-binding sites. The available evidence indicates that the mechanism of fatty acid synthesis is analogous to that previously described for the yeast and avian synthetases.
Δ9-Tetrahydrocannabinol–Protein Conjugates
Authors: Tsui P. T. , Kelly K. A. , Ponpipom M. M. , Strahilevitz M. , Sehon A. H.
Published: December 2009

The syntheses of four distinct derivatives of Δ9-tetrahydrocannabinol (Δ9-THC), intended for subsequent coupling to proteins and for use as hapten–protein conjugates for the production of anti-THC antibodies, are described. Three of these THC derivatives were coupled to proteins with the aid of the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and the fourth in the form of THC-isocyanate was conjugated directly to proteins.
The Effect of the Amount of Nucleic Acid Load on Hydroxylapatite Chromatography
Authors: Martinson H. G. , Wagenaar E. B.
Published: December 2009

The effect of the nucleic acid to hydroxylapatite load ratio (μg/g) has been studied. It was found that as the load ratio was increased from 1 to 100 μg/g there was little change in the elution molarity of the nucleic acids whereas above 1000 μg/g the change could be substantial. Different hydroxylapatite preparations and different nucleic acids gave similar results but the load ratio dependence of the elution molarity was greater at 70 °C than at room temperature.
Streptomycin Resistance and Ribosomal Proteins: Amino Acid Substitutions in the str Protein of One Streptomycin-Resistant Mutant of Escherichia coli K12 after Mutagenesis with Ethyl Methanesulfonate
Authors: Brakier-Gingras Léa , Lacoste Lucille , Boileau Guy
Published: December 2009

One streptomycin-resistant mutant of Escherichia coli K12 was selected after treatment with ethyl methanesulfonate. Comparison of tryptic peptide maps was used to investigate chemical changes in the str protein, S12. Two changes could be detected, which, from the comparison of the total amino acid composition, appear to result from the replacement of one tyrosine by one phenylalanine residue, and of one valine by one alanine residue. The observed amino acid substitutions differ from those found in protein S12 of previously investigated streptomycin-resistant mutants.
Biosynthèse "In Vitro" de l'Hormone Béta-Lipotropique de Boeuf
Authors: Bertagna X. , Lis M. , Gilardeau C. , Chrétien M.
Published: December 2009

Sheep beta-lipotropic hormone (β-LPH) is a pituitary hormone made of 90 amino acids and having a portion of its sequence (41–58) identical with the structure of beta-melanocyte-stimulating hormone (β-MSH). We hypothetized that β-LPH could be the biological precursor of β-MSH. We studied the biosynthesis of these two molecules by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated β-LPH from the other radioactive proteins with the usual method of purification described previously and we characterized the proteins by ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. Our results show that the pituitary slices synthesized a radioactive β-LPH which has all the characteristics of non-radioactive β-LPH. However, in the conditions used, we could not demonstrate any biosynthesis of β-MSH after 4 h incubation. These results suggest that the conversion of β-LPH into β-MSH, if it exists, is a slow process and should be studied in more prolonged incubations.
Identification and Distribution of p-Tyramine in the Rat
Authors: Philips S. R. , Durden D. A. , Boulton A. A.
Published: December 2009

A procedure for the quantitative evaluation of p-tyramine in mammalian tissues is described. It involves isolation of the amine by ion-exchange chromatography, followed by conversion to the dansyl derivative, chromatographic separation, and quantitation by the mass spectrometric integrated ion current technique using an isotopically labelled internal standard.The concentrations of p-tyramine in some tissues of male Wistar rats were (mean ± standard deviation (ng/g)): brain 2.0 ± 0.9, heart 3.4 ± 1.4, kidney 32.7 ± 13.1, liver 1.5 ± 0.5, lung 3.0 ± 1.2, and spleen 3.4 ± 1.2. In the brain, hypothalamus contained 11.3 ± 3.7, cerebellum 2.3 ± 1.7, stem 2.2 ± 1.0, caudate nucleus 19.2 ± 2.5, and the 'rest' 1.6 ± 0.4 ng/g, respectively.
Distribution, Metabolism, and Disappearance of Intraventricularly Injected p-Tyramine in the Rat
Authors: Wu P. H. , Boulton Alan A.
Published: December 2009

The major cerebral metabolic products of intraventricularly injected p-tyramine were octopamine and p-hydroxyphenylacetaldehyde. Only tiny amounts of p-hydroxyphenylacetic acid and p-hydroxyphenylethanol were found and these were located in the cerebral cortex (i.e. 'rest'). In brain regions isolated in the absence of pargyline the principal metabolite was p-hydroxyphenylacetaldehyde recovered in an acidic–neutral fraction; in the presence of pargyline, however, the majority of the label was associated with the amines tyramine and octopamine. In the caudate nucleus octopamine was essentially absent both in the presence and absence of pargyline.The loss of octopamine from all regions in the presence of pargyline was approximately exponential (i.e. first order) whereas in the absence of the monoamine oxidase inhibitor the loss was more complicated than first order. In most cases the loss of p-tyramine was greater than first order. Approximate half-lives based on these clearance values indicated a rapid turnover for p-tyramine and octopamine in all regions both in the presence and absence of pargyline.
Effect of Fatty Acyl Chain Length on Some Structural and Functional Parameters of Acholeplasma Membranes
Authors: Huang Leaf , Jaquet Donald D. , Haug Alfred
Published: December 2009

Two membrane preparations from Acholeplasma laidlawii were obtained by supplementing the growth media with either arachidic or lauric acid. The cells grown with arachidic acid supplementation yielded membrane lipids greatly enriched with the arachidoyl group and those supplemented with lauric acid yielded membrane lipids enriched with lauroyl, myristoyl, and palmitoyl groups. The cell size (0.1–0.7 μm), the membrane thickness (70 ± 14 Å), and the cell shape (coccoid) showed no difference for these two preparations. The arachidoyl-enriched membrane had a greater buoyant density (1.195 g/ml vs. 1.189 g/ml), a smaller permeability to glycerol, and a greater sensitivity to osmotic shock compared to the membrane enriched with shorter acyl groups. The spin label 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl is less mobile in the arachidoyl-enriched membrane (rotational correlation time at 22 °C, τc = 10 ns) than in the shorter acyl groups enriched membrane (τc = 6 ns). This difference in apparent membrane fluidity can account for the differences observed for the membrane properties.
Enzymatic Modifications of the Protamines. II. Separation and Characterization of Phosphorylated Species of Protamines from Trout Testis
Authors: Louie Andrew J. , Dixon Gordon H.
Published: December 2009

Substantial quantities of highly phosphorylated protamines were prepared from hormonally induced trout testes at the early protamine stage of development. Purified protamines from testes induced to mature by injection of salmon pituitary extracts were resolved into eight fractions on long carboxymethyl cellulose columns by eluting with a gradient of LiCl in the presence of 6 M urea; only two major fractions were found in protamine extracted from naturally maturing testes. Each fraction was not homogenous but consisted of a mixture of several related protamines. Analysis of radioactivity in in vivo32P-labeled protamine indicated that six of the eight fractions were phosphorylated. Amino acid analysis, phosphate determinations, and starch gel electrophoresis indicated that three of the phosphorylated peaks correspond to the mono-, di-, and triphosphorylated derivatives of the first fraction (three serines) of protamine, while the other three correspond to a similar series of the second fraction (four serines) of protamine. These protamines with differing levels of phosphorylation may be useful for in vitro studies of the interaction of phosphoprotamines with DNA or chromatin.
Regulatory Properties of Pyruvate Kinase from Liver of the Summer-Active Arctic Ground Squirrel
Authors: Behrisch Hans W. , Johnson Craig E.
Published: December 2009

The properties of liver pyruvate kinase (PyK) from summer-active Arctic ground squirrel (Citellus undulatus) were examined over the physiological temperature range of the animal. One form of the enzyme with a pI (isoelectric point) value of 5.3 was observed and exhibited kinetics similar to those of L-type PyK. This form has a molecular weight of 243 000, similar to that of M-type PyK. Enzyme–phosphoenolpyruvate (PEP) affinity falls with decreasing temperature while affinity for ADP increases under these conditions. The effect of temperature upon extent of negative cooperativity of PEP binding and its possible physiological importance are briefly discussed. Fructose-1,6-phosphate (FDP) increases the affinity of PyK for PEP but has no effect upon enzyme-ADP interaction. However, Vmax in the presence of saturating concentrations of both substrates is increased two to threefold by FDP, the allosteric activator. Ground squirrel PyK is inhibited by ATP. Inhibition by ATP of PyK activity is diminished by decreasing the temperature or by increasing the concentrations of substrate. FDP reverses this inhibition but the degree of reversal is sensitive to substrate concentration and temperature. Inhibition by ATP is competitive for the PEP site and appears to be of the "mixed-competitive" variety when carried out at varying concentrations of ADP. Inhibition by alanine is noncompetitive for both substrate sites. As a result of the very high concentrations of amino acids needed to obtain appreciable inhibition of live PyK, this does not appear to be a physiological means of regulation of enzyme activity. The effect of intermediates of the glycolytic, pentose shunt, and Krebs acid cycle pathways upon PyK activity is briefly discussed. Inosine nucleotides, involved in the reversal of the PyK reaction by phosphoenolpyruvate carboxykinase, have a strong modulating effect upon PyK activity. These data suggest formal mechanisms for the regulation of liver PyK in the ground squirrel.
Temperature Effect on the Permeability of Plasma Membranes of Advanced Germinal Cells of the Rat Testis
Authors: Lee Leo P. K.
Published: December 2009

Advanced germinal cells (spermatocytes and spermatids) prepared from adult rat testes, but not other cells, released their cellular contents into the incubation medium when they were incubated at 37° at a physiological pH. Evidence was presented that the release of cellular contents was due to changes in the permeability of the plasma membranes of these cells. The hypothesis is advanced that the changes in the permeability of the plasma membranes of the advanced testicular germinal cells of the rat may be intimately associated with the cellular degeneration and cessation of spermatogenesis which occur in cryptorchid testes.
The Synthesis of 2-Acetamido-2-deoxy-α-D-glucopyranose 4-Phosphate
Authors: Bundle David R. , Jennings Harold J.
Published: December 2009

The synthesis of 2-acetamido-2-deoxy-D-glucose 4-phosphate is described. A suitably protected derivative (benzyl 2-acetamido-3-O-acetyl-6-O-benzoyl-2-deoxy-β-D-glucopyranoside) was phosphorylated with o-phenylene phosphochloridate. The removal of the o-hydroxyphenyl group was accomplished by using 2% bromine in buffered aqueous-ethanol, and the ester groups were removed by mild alkaline treatment. Removal of the benzyl glycosidic group by hydrogenation yielded 2-acetamido-2-deoxy-D-glucose 4-phosphate, which was obtained in a crystalline form as its monoammonium salt monohydrate.
Time Differential in Labelling of Different Monosaccharides into Serum Lipoproteins in Rats
Authors: Mookerjea Sailen , Miller Carole
Published: December 2009

A study on the time-course of incorporation of radioactive galactose, fucose, glucosamine, mannose, and leucine into serum lipoproteins in vivo showed that the incorporation of fucose and galactose in the lipoproteins reached a peak within 30–60 min, whereas that for glucosamine and leucine was not reached until 120 min. As newly synthesized lipoproteins have to traverse through rough and smooth endoplasmic reticulum, Golgi, and plasma membranes, it is expected that the sugars that are incorporated in the oligosaccharide chain in the rough microsomes should be secreted out into the serum at a later time than the sugars that are primarily incorporated at the Golgi membranes. The difference in time of incorporation of glucosamine vs. galactose and fucose seems to be in agreement with this expectation. Leucine is incorporated in the membrane-bound ribosomes and its peak incorporation into serum lipoproteins was, therefore, not reached until 120 min. Incorporation of mannose into lipoproteins showed biphasic pattern indicating its possible metabolic conversion into other sugars. Apolipoproteins labelled with fucose and galactose showed about 66–94% recovery of the respective labels after acid hydrolysis and chromatographic separation.
Control of RNA Synthesis by Amino Acids in Landschutz Ascites Tumor Cells
Authors: Jolicoeur Paul , Labrie Fernand
Published: December 2009

Landschutz cells incubated in amino acid-deficient medium for 2.5 h show a markedly reduced incorporation of [3H]uridine into 18 S and 28 S cytoplasmic ribosomal RNA (rRNA) and into 28 S, 32 S, and 36 S nuclear RNA measured during the last 90 min of incubation, whereas the radioactivity associated with 45 S pre-rRNA is not affected. Ten-minute pulse-labeling and 15-min pulse-chase experiments show that amino acid starvation inhibits both the synthesis and processing of 45 S pre-rRNA. Amino acid starvation has no significant effect on the labeling of the nucleotide pools. This effect of amino acids was specific for rRNA since the synthesis of 4 S and 5 S cytoplasmic RNA separated on polyacrylamide gels and of polysomal mRNA analyzed on sucrose gradients was not significantly affected during amino acid starvation. These data also indicate that RNA synthesis is non-coordinated in Landschutz cells. Among the 13 amino acids essential for growth of these cells, arginine and glutamine appear to be mainly responsible for the inhibition of synthesis of 18 S and 28 S rRNA measured during incubation in complete amino acid-deficient medium. The removal of any one of the other amino acids has a small inhibitory effect on the incorporation of [3H]uridine into rRNA and their effect on the synthesis of 18 S rRNA is more pronounced than on that of 28 S rRNA. Such effect results in an unbalanced production of these two ribosomal RNA species.
Temperature and the Regulation of Enzyme Activity in the Hibernator. Isoenzymes of Liver Pyruvate Kinase from the Hibernating and Non-hibernating Arctic Ground Squirrel
Authors: Behrisch Hans W.
Published: December 2009

Liver of the hibernating (H) Arctic ground squirrel (Citellus undulatus) contains a single species of pyruvate kinase (PyK) that is distinct from the single isoenzyme of pyK observed in the non-hibernating (NH) ground squirrel, which has been previously described (Behrisch &Johnson (1974) Can. J. Biochem. 52, 547–559). The H-PyK has a pI value of 5.7 and a molecular weight of 241 000 – 243 000. Affinity of the H-PyK for the substrates phosphoenolpyruvate (PEP) and ADP is not affected by changing temperature. It is argued that this stability of the apparent Km's for substrate over a wide temperature range permits the hibernator to take advantage of the Q10 effect in maintaining a low rate of the PyK reaction. Similarly, affinity of H-PyK for the allosteric activator fructose-1,6-phosphate (FDP) and the inhibitor ATP is also conspicuously independent of temperature, suggesting a fine stoichiometry in the relative concentrations of the regulatory ligands in control of H-PyK over a wide temperature range. Further, affinity of H-PyK for the inhibitor ATP is about three- to fourfold lower than that of the NH-PyK, a condition that would favor the maintenance of a high energy charge in the hibernating liver cell. ATP apparently inhibits PyK by causing a dissociation of the enzyme molecule into two "halves" of about 110 000 molecular weight each. This dissociation is offset and reversed by FDP. Removal of the ATP by dialysis does not of itself result in a reassociation of the PyK "halves"; FDP and/or the substrates are required for the two subunits of PyK to reassociate. As the apparent Ki of H-PyK for ATP is higher than that of NH-PyK, substantially higher concentrations of ATP are required to effect the dissociation of H-PyK. Similarly, elevated concentrations of FDP are required to offset the ATP-caused dissociation of the H-PyK.Hibernating Arctic ground squirrels that are preparing to emerge finally from the hibernating state already possess substantial activities of the NH-PyK isoenzyme. This suggests that the animal "anticipates" its transition from one metabolic state from another. On the basis of these data a formal mechanism is proposed for the regulation of liver PyK in the Arctic ground squirrel in both the non-hibernating and hibernating states.
The Purification and Properties of an Amidohydrolase from Soybean
Authors: Hoagland Robert E. , Graf George
Published: December 2009

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.
The RNA Fractions of Chromatin from L-Cells
Authors: Monahan John J. , Hall Ross H.
Published: December 2009

Chromatin obtained from L-cell nuclei has been shown to contain three distinct RNA fractions. One, a low molecular weight RNA fraction (3–7 S), has been isolated and purified. Two-dimensional polyacrylamide gel electrophoresis indicates that this fraction contains at least 28 diverse RNA components. Both the rate of incorporation of [32P]H3PO4 into this fraction and the loss of [3H]uridine from the fraction after a 1 h pulse suggest that these RNA's are slowly synthesized and are very stable. Not all the species within the low molecular weight fraction have the same turnover rates.A high molecular weight RNA fraction (15–30 S) has also been isolated from L-cell chromatin. From the rate of [32P]H3PO4 incorporation and the loss of [3H]uridine from this fraction after a 1 h pulse, it appears that this RNA fraction is rapidly synthesized and is either unstable or is rapidly removed from chromatin once synthesized.A third RNA fraction also present in L-cell chromatin appears to be tightly bound to the DNA. It has been isolated and shown to be of low molecular weight (3–5 S). This RNA also appears to be rapidly synthesized and to be metabolically unstable.
Physiochemical Properties of Thermomycolase, the Thermostable, Extracellular, Serine Protease of the Fungus Malbranchea pulchella
Authors: Voordouw Gerrit , Gaucher G. Maurice , Roche Rodney S.
Published: December 2009

The physicochemical properties of the extracellular protease of the fungus Malbranchea pulchella, for which we have adopted the name thermomycolase, were investigated. The molecular weight of diisopropylphosphorylthermomycolase was found to be 32 000–33 000 by sedimentation equilibrium and sodium dodecyl sulfate (SDS) gel electrophoresis. Its sedimentation coefficient (s020, w = 2.97 S), intrinsic viscosity ([η] = 3.0 cc/g), and frictional ratio (f/f0 = 1.09) characterize the enzyme as a typical globular protein. The circular dichroism spectrum of the protein is also consistent with its globular structure. Active thermomycolase autolyzes extensively, especially at low calcium ion concentrations, producing low molecular weight peptide material. In the presence of SDS, a different autolytic degradation is observed, resulting in much higher molecular weight polypeptide products (16 500, 12 500,11 000, 8 500). The results indicate that, in the presence of SDS, thermomycolase has three sites that are highly susceptible to autolysis. At calcium ion concentrations of 10−3 M and 10−2 M the enzyme undergoes a sharp thermal denaturation with transition temperatures at 69 °C and 75 °C, respectively, and with complete loss of enzyme activity. At 70 °C the enzyme appeared to be maximally thermostable at a calcium ion concentration of 10−2 M.
Diagonal Chromatography for the Selective Purification of Tyrosyl Peptides
Authors: Cruickshank William H. , Malchy Barry L. , Kaplan Harvey
Published: December 2009

Thiolysis of an O-dinitrophenyl-tyrosyl peptide results in an increased solubility in the stationary phase of a n-butanol – acetic acid – water – pyridine (15:3:12:10) (BAWP) paper chromatographic system. It is shown that this property can be used to form the basis of a diagonal paper chromatographic purification of tyrosyl peptides from enzymatic digests of proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is subjected to enzymatic digestion, applied to a strip of Whatman 3 MM paper, thiolyzed with 5% 2-mercaptoethanol in acetone, and subjected to chromatography using BAWP as solvent. A guide strip is removed, thiolyzed with 5% 2-mercaptoethanol in 25% pyridine, and resubjected to chromatography in BAWP at right angles to the original direction of chromatography. The tyrosyl peptides are displaced off the diagonal towards the origin. The off-diagonal peptides are isolated from the original chromatogram by thiolysis and chromatography using the diagonal chromatogram to locate the positions of the dinitrophenyl-tyrosyl peptides.
Wide-Angle X-Ray-Diffraction Studies of the Bovine-Erythrocyte Membrane. Treatment with Sodium Chloride, Deoxycholate, and Lipid Solvents
Authors: Dresdner George W. , Cid-Dresdner Hilda , Meza Luis , Cardemil Emilio
Published: December 2009

X-ray diffraction studies have been carried out on treated and untreated bovine-erythrocyte membranes. Treatment was with 0.8 M NaCl, 0.5% sodium deoxycholate, and lipid solvents. The following spacings have been identified: 2.13 Å, 3.34 Å, 4.34 Å, 8.5–10.5 Å, and 43 Å (1 Å = 10−10 m). The 2.13-Å spacing does not seem to be characteristic of any of the membrane components but seems to represent a paraffin-like liquid phase in the membrane. The 3.34-Å spacing corresponds to a diffraction ring of water and protein. The 4.34-Å spacing seems to be produced by membrane protein and lipid, the 8.5–10.5-Å spacing by protein, and the 43-Å spacing by phospholipid. The results suggest the presence of zones where protein and lipid have an arrangement independent of each other in the membrane, and suggest that at least some of the protein could be of the fibrous type.
Purification of Escherichia coli Chloramphenicol Acetyltransferase by Affinity Chromatography
Authors: Guitard Michel , Daigneault Réjean
Published: December 2009

Chloramphenicol acetyltransferase (CATase) was purified by affinity chromatography from Escherichia coli W677/HJR66, an R-factor-bearing mutant. The chloramphenicol aryl nitro group had to be reduced to an amino group prior to its coupling to the Sepharose 4B matrix. The single-step isolation procedure resulted in a 237-fold purification of CATase with over 65% recovery of the enzyme.
Studies on the Mechanism and Reversal of the Phospholipase-A2 Inactivation of D-Glucose Uptake by Isolated Human Erythrocyte Membranes
Authors: Banjo Batya , Walker Caroline , Rohrlick Ruth , Kahlenberg Arthur
Published: December 2009

The mechanism underlying the inactivation of the stereospecific uptake of D-glucose by isolated human-erythrocyte membranes following digestion with phospholipase A2 (Kahlenberg, A. &Banjo, B. (1972) J. Biol. Chem 247, 1156–1160) was investigated. This inactivation was not accompanied by any significant change in the uptake of L-glucose. The decrease in D-glucose uptake following limited (25–30%) cleavage of membrane phospholipids by phospholipase A2 was characterized by a twofold increase in the apparent dissociation constant of the D-glucose–membrane complex and a 34% decrease in the membrane's maximum capacity for D-glucose uptake. These effects of phospholipase A2 were completely reversed upon removal of the membrane-bound phospholipid byproducts (fatty acids and lysophospholipids) by washing the membranes with defatted bovine-serum albumin. Oleic acid and various lysophosphatides added to albumin-washed, phospholipase A2-treated membranes in amounts formed by the enzyme treatment produced negligible inhibition of D-glucose uptake. With more extensive phospholipase A2 digestion of membrane phospholipids, defatted bovine-serum albumin did not restore D-glucose uptake despite the removal of the phospholipid by-products formed.In addition to the inactivation of D-glucose uptake, limited enzyme treatment transforms the appearance of the membranes collected by centrifugation from opaque white to transparent and gelatinous. Both of these effects of phospholipase A2 are completely reversed upon incubation of the membranes at pH 5.5 for 2 h at 37 °C without loss of any of the membrane lysophosphatides and fatty acids formed by the enzyme treatment. It is suggested that this pH- and temperature-dependent restoration of D-glucose uptake is due to a conformational change resulting in the relocation of the membrane D-glucose-binding sites into a functional environment.These results indicate that the inactivation of D-glucose uptake by phospholipase A2, which was not accompanied by any change in L-glucose uptake, occurs by two different mechanisms. With limited hydrolysis of membrane phospholipids, one or both of the resulting phospholipid by-products reversibly inhibit the uptake of D-glucose by decreasing the affinity of the membrane for D-glucose and by masking a portion of the total available D-glucose-binding sites. However, upon extensive cleavage of phospholipids in the hydrophobic region of the membrane, there is an apparently irreversible disorganization of the membrane D-glucose-binding component. This might be due to destruction of vital phospholipids and/or a disturbance of the interactions between the lipid and protein components of the membrane.
Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods
Authors: Suria D. , Liew C. C.
Published: December 2009

Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.
Selective Labelling of the Methyl Carboxylate Substituents Found in the Anticodon Sequences of Some Species of Yeast Transfer RNA
Authors: Kennedy T. D. , Lane B. G.
Published: December 2009

(1) By incubation in 0.1 M NaOH for 10 min at room temperature, it is possible to "saponify" some of the methyl carboxylate linkages in bulk yeast tRNA. By incubation with S-adenosyl[Me-14C]methionine and either homologous (yeast) or heterologous (wheat-embryo) enzymes, it is then possible to "re-esterify" the "saponified" tRNA and thereby effect selective labelling at 5-carboxymethyluridine [Me-14C]methyl ester residues. (2) There is also selective labelling at 2-thio-5-carboxymethyluridine [Me-14C]methyl ester residues when "saponified" yeast tRNA is incubated with S-adenosyl[Me-14C]methionine and homologous (but not heterologous) enzymes. (3) When selectively labelled yeast tRNA is hydrolyzed by RNase T1, both 5-carboxymethyluridine [Me-14C]methyl ester and its 2-thio-analogue are released as part of large oligonucleotides, each of which contains roughly 10 nucleotide residues. (4) There are at least three, and possibly four [Me-14C]-methyl ester-containing oligonucleotides released by RNase T1 digestion of selectively labelled "saponified" yeast tRNA. A comparison of the chromatographic properties of the different [Me-14C]oligonucleotides suggests that the same 5-carboxymethyluridine residues are probably targets for both homologous and heterologous enzymes. (5) The properties of the selectively labelled oligonucleotides are consistent with the view that some of them probably are derived from yeast tRNA3Glu, tRNA2Lys, and tRNA3Arg, ail of which are known to contain 5-carboxymethyl methyl esters as part of their anticodon sequences.
Synthesis and Some Biological Properties of 1-Deamino-4-Glu-Oxytocin (1-β-Mercaptopropionic Acid–4-Glutamic Acid–Oxytocin) and its Use in Preparing a Hormone–Agarose Complex
Authors: Ferrier B. M. , Branda L. A.
Published: December 2009

1-Deamino-4-Glu-oxytocin (1-β-mercaptopropionic acid – 4-glutamic acid – oxytocin) was synthesized by sequential reduction by sodium in liquid ammonia and oxidation by hydrogen peroxide of the octapeptide derivative, S-benzyl-β-mercaptopropionyl-tyrosyl-isoleucyl-γ-O-benzyl-glutamyl-asparaginyl-S-benzyl-cysteinyl-prolyl-leucyl-glycinamide. The oxidation analogue was isolated and purified by partition chromatography in two different solvent systems followed by exclusion chromatography on Sephadex G-25. It was found to possess approximately 13 I.U. of uterotonic activity, 34 I.U. of milk ejection activity, and 83 I.U. of milk ejection-like activity per milligram, measured on an isolated strip of lactating mouse mammary gland. 1-Deamino-4-Glu-oxytocin was coupled to AH-Sepharose 4B by way of the free γ-carboxyl group of its residue of glutamic acid. The water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride caused the coupling with approximately 70% effectiveness. The resultant peptide–agarose complex had low biological potency in the assay of milk ejection-like activity.