bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on Protein Subunits. V. Specific Interaction between Matrix–Bound Subunits of Aldolase and Soluble Aldolase Subunits
Authors: Chan W. W.-C.
Published: December 2009

A derivative containing matrix-bound (MB) subunits of aldolase was prepared by attaching native tetrameric aldolase to Sepharose to form MB-aldolase followed by dissociation with 6 M guanidinium chloride and renaturation. The interaction between the MB-subunit aldolase derivative so prepared and subunits added in solution was studied. Nascent subunits of aldolase were generated in situ by diluting a small aliquot of guanidinium chloride-denatured aldolase into a much larger volume of buffer containing a suspension of MB-subunit aldolase. This treatment caused a significant increase in the amount of bound activity. The ability of MB-subunit aldolase to pick up activity from solution is highly specific. After repeated treatments with nascent sub-units, the matrix-bound activity reached a saturation level close to four times the activity of MB-subunit aldolase. The product of this treatment is very similar in properties to the original MB-tetrameric aldolase but different from MB-subunit aldolase. These observations suggest that MB-subunit aldolase can associate with nascent subunits generated in solution to form MB-tetrameric aldolase. The results in this paper support the conclusion from the kinetics of renaturation (Chan et al. J. Biol. Chem. 248, 2778 (1973)) that aldolase monomers have the same activity whether they exist singly or as part of a tetrameric structure.
Studies on Acute Phase Proteins of Rat Serum. IV. Pathway of Secretion of Albumin and α1-Acid Glycoprotein from Liver
Authors: Jamieson J. C. , Ashton F. E.
Published: December 2009

Normal rats and those suffering from inflammation for 12 h were given pulse injections of L-leucine-3H and D-glucosamine-14C and were killed at 3–60 min thereafter. Rough and smooth microsome fractions and a Golgi-enriched fraction were prepared from liver and the fractions were extracted with the non-ionic detergent Lubrol-W. Albumin and acute phase α1-acid glycoprotein were isolated from the extracts by application of a quantitative precipitin technique employing antisera to albumin and α1-acid glycoprotein, and specific radioactivities were determined. The results indicated that the pathway of secretion of albumin and α1-acid glycoprotein was rough endoplasmic reticulum → smooth endoplasmic reticulum → Golgi complex → blood. Although there was little difference in the pathway or rate of secretion of the proteins under study in normal and experimental animals, there was an increase in specific radioactivities of labelled leucine and glucosamine associated with α1-acid glycoprotein when isolated from subcellular fractions from livers from experimental animals.
Phosphorylation of Histones in Avian Erythroblasts
Authors: Seligy V. L. , Neelin J. M.
Published: December 2009

Incorporation of 32P-orthophosphate into erythrocyte histones, purified by the methods of Amberlite CG-50 cation-exchange and Bio-Gel P-60 exclusion chromatography, has been studied on three cell populations of avian blood, enriched in mature cells, reticulocytes, and erythroblasts, respectively. Phosphate uptake in the histones was differential; the order of specific activities was as follows: IIb1 > V > IV >I > IIb2 > III. Incorporation into all histone fractions was considerably greater in erythroblast-enriched blood than in reticulocyte-enriched blood, which incorporated only slightly more 32P-phosphate into histones than did normal blood cells.The tryptic peptides from the respective 32P-histones obtained from each cell population did not differ significantly. Both histone I and V had multiple bands of 32P-phosphopeptides, which were similar, but not identical, in ionophoretic mobilities; in contrast, IIb1 and IV gave only a single major band each with identical ionophoretic mobilities. Much of the 32P-phosphate in IIb2 and III appeared to arise from contaminating IIb1 and IV–V, respectively.
On the Substrate Specificity of Jack Bean Urease (Urea amidohydrolase, EC 3.5.1.5)
Authors: Gazzola Carlo , Blakeley Robert L. , Zerner Burt
Published: December 2009

pH-Stat studies prove that N-methylurea, ethyl carbamate, and carbamoyl azide are not substrates for jack bean urease. Semicarbazide is a good substrate, showing Michaelis–Menten kinetics and stoichiometric formation of ammonia, carbonic acid, and hydrazine, and having a kcat of about [Formula: see text] that of urea at pH 7.
The Chemical Composition and Serological Reactions of Lipopolysaccharides from Serogroups A, B, X, and Y Neisseria meningitidis
Authors: Jennings H. J. , Hawes G. B. , Adams G. A. , Kenny C. P.
Published: December 2009

Analyses of the cell wall lipopolysaccharides prepared from Neisseria meningitidis serogroups A, B, X, and Y, indicated that they all contained glucose, galactose, glucosamine, heptose, lipid A, ethanolamine, fatty acids, phosphate, and protein. Some minor compositional differences in the sugar components did occur in that sialic acid (6–7%) was found only in serogroups B and Y, and galactosamine (2–3%) in serogroups B and X. The individual fatty acid and amino acid components in the four serogroups were also qualitatively similar. The Neisseria lipopolysaccharides studied have the same components as the core structure of the Enterobacterioceae but appear to lack the characteristic components of their O-antigen side chains. Serological studies indicated that the lipopolysaccharides were homogeneous and were for the main part group specific in nature, despite their compositional similarity. On the basis of compositional differences alone it is not possible to account for the group specificity that they exhibit.
Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. II. Kinetic Properties
Authors: Wu Tai-Wing , Scrimgeour K. G.
Published: December 2009

Some of the kinetic properties of inosine 5′-phosphate (IMP) dehydrogenase purified from Bacillus subtilis have been described. K+ can be replaced as an activator by other monovalent cations, whose activities are related to their ionic radii. IMP dehydrogenase is stimulated by appropriate concentrations of adenosine 5′-phosphate, and is strongly inhibited by the antibiotic mycophenolic acid. The enzyme is inhibited by guanosine 5′-phosphate (GMP), a suspected negative feedback inhibitor, in a complex manner. Desensitization of this inhibition suggests that GMP binds at a site distinct from the catalytic site. Several properties of the enzyme change after desensitization. For example, the pH optimum shifts from pH 7.55 to pH 8.4, and whereas the inhibition by GMP is greatly reduced there is an increase in catalytic activity. These studies and the oligomeric structure of IMP dehydrogenase strongly indicate a regulatory role for the enzyme in B. subtilis.
Biosynthesis of Vitamin B6. Incorporation of Terminally Labelled Glucose
Authors: Hill Robert E. , Spenser Ian D.
Published: December 2009

Radioactivity from D-[1-14C]glucose as well as from D-[6-14C]glucose is confined to C-2′, -4′, and -5′ of pyridoxol, but the quantitative distribution of label among these three carbon atoms is different in the two cases. These results are interpreted in terms of the early stages of the biosynthesis of vitamin B6.
Adenine Reversal of Ethionine-Induced Inhibition of Protein Synthesis: A Comparison with Computer-Generated Data
Authors: Kisilevsky R. , Matheson G.
Published: December 2009

In vivo labeling kinetics of (a) nascent peptides on liver polysomes, and (b) proteins of the 100 000 × g supernatant have been determined for ethionine-intoxicated female rats given adenine, to reverse the effect of ethionine. When compared to computer-generated data the results indicate that the reversal effect of adenine is not simply through the increase in the previously reduced rate of initiation. In addition to increasing the rate of initiation, adenine, soon after its administration, reduces the rate of elongation. The rate of elongation subsequently returns to normal levels.
Structure of the Ribosome: Exposure of Ribosomal Proteins Determined by Competitive Labelling
Authors: Visentin L. P. , Yaguchi M. , Kaplan H.
Published: December 2009

Reactivities of ribosomal proteins toward acetic anhydride as determined by competitive labelling give numerical estimates of their exposure and deflect the interrelationships in the macrostructure.
Electrophoretic Studies of Three Variants of Mengo Encephalomyelitis Virus
Authors: Chlumecka Vera , D'Obrenan P. , Colter J. S.
Published: December 2009

A number of earlier studies of three plaque variants of Mengo virus (designated L-, M-, and S-Mengo), all of which suggested that they differ from one another with respect to surface charge characteristics, prompted an examination of the variants by the technique of isoelectric focusing. Highly purified, radiolabelled virus preparations were subjected to electrofocusing in sucrose-stabilized, pH gradients in the presence of 0.5% Brij 35. The distribution of each variant was found to be bimodal; the average values for the pH's at which the major (75–90% of input virions) and minor peaks were found were as follows: L-Mengo, 8.1 and 4.6; M-Mengo, 4.4 and 6.3; S-Mengo, 4.6 and 6.8 Infectivity assays established that the distributions of plaque-forming units, (p.f.u.) and radioactivity were the same (c.p.m./p.f.u. ratios in the major and minor peaks were essentially identical), and re-electrofocusing of virions from each peak was found to give the same bimodal distribution as did the original preparation. The M- and S- variants were found to migrate more rapidly than L-Mengo during electrophoresis in polyacrylamide gels at pH 8.3. The data arising from the electrofocusing studies are compatible with observations regarding the relative rates of attachment of L-, M-, and S-Mengo to cultured L cells and their relative abilities to hemagglutinate human erythrocytes (type O) at pH 8.0.
Inhibition of Aminoacyl-tRNA Synthetases From Rat Liver by Reducing Agents
Authors: Vargas Rocío , Castaneda Mario
Published: December 2009

Commonly used reducing agents 2-mercaptoethanol and dithiothreitol inhibit in vitro homologous aminoacylation of rat liver systems. The extent of inhibition depends on the buffer and is greater in Tris than in N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES). Variations in the values of pH and the concentrations of Mg2+, ATP, tRNA, enzyme preparation, and buffers were ineffective to reverse the inhibition produced by these reducing agents. Enzyme activity with no reducing agent is, in general, the same or higher in Tris than in HEPES, although it depends on the pH values and the concentrations of buffer and Mg2+ in the reaction mixture.
The Role of the Mitochondrial Citrate Transporter in the Regulation of Fatty Acid Synthesis: Effect of Fasting and Diabetes
Authors: Cheema-Dhadli Surinder , Halperin Mitchell L.
Published: December 2009

The Michaelis constant (Km) for citrate for the hepatic mitochondrial citrate transporter of fed rats is 239 μM. This Km increases approximately twofold in mitochondria of fasted or diabetic rats. The long-chain fatty acyl-CoA content was approximately twofold higher in mitochondria from fasted and diabetic rats and could account for this increased Km. The role of regulation of the mitochondrial citrate transporter in lipogenesis is discussed.
Activation of Pyruvate Dehydrogenase in Adipose Tissue: Role of Insulin and Pyruvate
Authors: Berman Bruce G. , Halperin Mitchell L.
Published: December 2009

This study was designed to investigate the relation between activation of pyruvate dehydrogenase in white adipose tissue and the intracellular pyruvate content. The intracellular pyruvate level was approximately 8 nmol/g wet weight when adipose tissue was incubated up to 20 min with fructose (1 mg/ml) as substrate. The addition of insulin to the incubation medium resulted in a 1.5-fold increase in the active form of pyruvate dehydrogenase after 20 min incubation without raising the intracellular pyruvate level at earlier time periods. Intracellular pyruvate contents were increased 1.5-fold by TMPD (75 μM), but in this case there was no concomitant activation of pyruvate dehydrogenase. We conclude that the insulin-induced activation of pyruvate dehydrogenase is caused by mechanism(s) in addition to an elevation of pyruvate concentration.
Partial Sequence of Trypsin Inhibitor From Ovomucoid
Authors: Murthy G. S. , Gilardeau C. , Chrétien M.
Published: December 2009

The amino acid sequence of the 40 amino acid residues at the N-terminal region of trypsin inhibitor from chick ovomucoid has been determined. This part of the sequence does not show any homology with any region of the other known trypsin inhibitors.
Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase
Authors: Seah Tony C. M. , Bhatti A. R. , Kaplan J. G.
Published: December 2009

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.
Thiol–Protein Disulfide Oxidoreductase Activity in Lactating and Non-lactating Mouse Mammary Gland
Authors: Ferrier B. M. , Johns T. , Say A. , Branda L. A.
Published: December 2009

Glutathione–protein disulfide oxidoreductase activity has been demonstrated in mouse mammary gland, with both insulin and oxytocin serving as disulfide-containing substrates. The activity does not change markedly in pregnancy but shows a marked increase when lactation is established, reaching a maximum about the 10th day of lactation.
Metabolite Synthesis by Rat Liver Cells and Rat Liver Mitochondria
Authors: Sauer F. , Mahadevan S.
Published: December 2009

The use of rat liver cells prepared by mechanical dispersion techniques has recently been severely criticized on the basis that resulting plasma membrane damage leads to total loss of cytoplasmic constituents, thus leaving only membrane sacs containing mitochondria. In the present study, metabolite synthesis by mechanically prepared cells (M cells), collagenase prepared cells (E cells), and isolated mitochondria is compared.It was found that M cells have high rates of endogenous fatty acid oxidation and incorporate [1-14C]acetate approximately equally into 3-hydroxybutyrate, glutamate, citrate, malate, and 2-oxoglutarate. In contrast, mitochondria synthesize primarily citrate and aspartate but no 3-hydroxybutyrate from [1-14C]acetate. Loss of some cytoplasmic enzymes from M cells during isolation was confirmed. M cells are incapable of gluconeogenesis from lactate, synthesize much less glutamate than E cells, and become more highly oxidized during incubation ([NAD+]/[NADH] ratio of 135 versus 30 for E cells). Metabolite synthesis from [1-14C]acetate by mitochondria can be regulated by adding very low concentrations of organic acids and electron donors to the incubation.From these results it was concluded that M cells carry out metabolic reactions not present in isolated mitochondria. Metabolic reactions in mitochondria will approximate those of M cells if electron donors and mixtures of anions are added to the incubation.
Biosynthesis of Dipalmitoyllecithin by the Rabbit Lung
Authors: Kyei-Aboagye K. , Rubinstein D. , Beck J. C.
Published: December 2009

The esterification of palmitate by lung homogenates is increased by palmitoyllysolecithin in the presence of CoA and ATP. [32P]lysolecithin can also be converted to [32P]lecithin in the absence of other substrates, suggesting transacylation between two molecules of palmitoyllysolecithin. Some palmitate is esterified in the absence of added lysolecithin, ATP, and CoA. Free palmitate appears to exchange with other esterified fatty acids, especially oleate incorporated into the lecithin of lung tissue slices. The lung contains sufficient 1-palmitoyl-2-oleyllecithin for this exchange to be significant. In homogenates, Ca2+ is required for the exchange and the incorporation of palmitate. The in vivo turnover of the oleate in the lecithin of lung tissues and bronchiolar washings is more rapid than that of the palmitate. This is consistent with a mechanism of dipalmitoyllecithin synthesis involving exchange of esterified oleate for palmitate in lecithin. It is concluded that in addition to de novo synthesis of lecithin in the lung, incorporation of activated palmitate into lysolecithin and exchange of palmitate with oleate esterified in endogenous lecithin also occur. Phospholipase activity is probably involved in the latter two pathways.
Studies of Folic Acid Compounds in Nature. III. Folic Acid Compounds in Cabbage
Authors: Chan C. , Shin Y. S. , Stokstad E. L. R.
Published: December 2009

The folate pattern in cabbage was studied by chromatography on Sephadex G-25 and DEAE-cellulose columns associated with markers of high specific radioactivity and with differential microbiological assay. More than 90% of cabbage folate is polyglutamates containing more than five glutamic acid residues, mainly as the 5-methyl derivatives. The distribution is as follows: 52% of total cabbage folate is 5-CH3-H4PteGlu7, 32% is 5-CH3-H4PteGluc6, 7% is 5-CH3-H4PteGlu8, 4% is 5-CHO-H4PteGlu7, and the rest is 5-CH3-H4PteGlu3–5.
The Biosyethetic Origin of Chlorflavonin, a Flavonoid Antibiotic from Aspergillus candidus
Authors: Marchelli R. , Vining L. C.
Published: December 2009

Cultures of Aspergillus candidus produce, in addition to chlorflavonin, a closely related metabolite that has been isolated and characterized as the dechloro analogue. Radioactivity from [14C]dechlorochlorflavonin was incorporated irreversibly into chlorflavonin. Cultures supplemented with D-[U-14C]glucose, L-[methyl-14C]methionine, L-[U-14C]phenylalanine, or L-[β-14C]-phenylalanine yielded the 14C-labeled metabolites; chemical degradation of chlorflavonin labeled by the last named precursor showed that the isotope had been incorporated specifically into position 2 of the flavonoid carbon skeleton. We conclude that chlorflavonin is synthesized de novo by A. candidus and is, therefore, the first proven fungal flavonoid.
Acid Proteases From Species of Mucor: Molecular Weight of Mucor miehei Protease From Amino Acid Analysis Data
Authors: Rickert W. S. , Elliott J. R.
Published: December 2009

An improved method for the isolation of Mucor miehei protease which utilizes a diafiltration cell has been used to obtain a highly purified protein in gram quantities and yields of about 80%. Based on a modified molecular weight function and data from amino acid analysis, a value of 41 800 for the molecular weight of the glycoprotein was established and some modification to the published amino acid composition was made. These results suggest that Mucor miehei protease is distinctly different from the two other acid proteases which are also produced by species of Mucor.
Formation of Estrogen Glucosiduronates by Human Kidney Homogenates
Authors: Hobkirk R. , Green R. N. , Nilsen M. , Jennings B. A.
Published: December 2009

17β-Estradiol-6,7-3H (E2), 17β-estradiol-6,7-3H-3-sulfate (E23S), and estriol-6,7-3H (E3) were each incubated with human kidney homogenates in the presence of uridine diphosphoglucuronic acid. Metabolites were purified by DEAE-Sephadex and Celite partition chromatography and were identified by crystallization with carrier steroid conjugates and free steroids. E2 was converted to a small but definite extent (< 0.1–5%) to estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, and 17β-estradiol-17-glucosiduronate, the latter conjugate usually predominating. Under the experimental conditions E2 was a better precursor of all three conjugates than was E23S. In one experiment where kidney cortex and medulla were incubated separately with E2, the former was some 20 times more efficient in glucosiduronate synthesis. E3 was converted to the extent of 52–91% to estriol-16-glucosiduronate by whole kidney homogenates.
Evaluation of Three Constants Involved in the Binding of Corticosterone to Plasma Proteins in the Rat
Authors: Jobin Michel , Perrin François
Published: December 2009

Equilibrium dialysis was used to evaluate three constants involved in the binding of corticosterone to transcortin and albumin in rat plasma, namely ST (concentration of binding sites of transcortin), KT (association constant of transcortin), and SAKA (binding constant of albumin). Undiluted plasma from rats adrenalectomized 24 h earlier was enriched with increasing amounts of corticosterone (0–5.0 μg/ml) and dialyzed for 4.3 h at 37 °C.Assuming that the law of mass action applies to the interaction between corticosterone and its two binding proteins, the values of ST, KT, and SAKA were estimated by means of a new method of calculation. Derived from the maximum likelihood principle, this method allows computation of a confidence region for the parameters. The following values and statistical bounds were obtained:[Formula: see text]
The Separation and Partial Purification of the Soluble 17α- and 17β-Hydroxysteroid Dehydrogenases of Rabbit Liver
Authors: Hasnain Sadiq , Williamson Denis G.
Published: December 2009

17-Hydroxysteroid dehydrogenase activity toward 17α-estradiol, 17β-estradiol, and the 3-glucuronide derivatives of these steroids has been demonstrated in the 105 000 × g supernatant of rabbit liver. DEAE-cellulose chromatography of the enzyme activities isolated by calcium phosphate gel fractionation and Sephadex gel filtration yielded a single 17α-hydroxysteroid dehydrogenase activity toward both 17α-estradiol and 17α-estradiol 3-glucuronide. However, separate 17β-hydroxysteroid dehydrogenase activities toward 17β-estradiol and 17β-estradiol 3-glucuronide were obtained. Further purification of the 17α-hydroxysteroid dehydrogenase fraction by isoelectric focusing resulted in multiple peaks of activity toward 17α-estradiol and 17α-estradiol 3-glucuronide. One of these enzyme activities was highly specific for the 3-glucuronide derivative, it being essentially devoid of activity toward 17α-estradiol.
Catalysis of Exchange of Oxygen Into and Out of L-Leucine by Leucine Aminopeptidase
Authors: Wells James A. , Glasser Gary L. , Carpenter Frederick H.
Published: December 2009

Leucine aminopeptidase (L-leucyl-peptide hydrolase EC 3.4.1.1) was shown to catalyze the exchange of oxygen-18 into and out of L-leucine. Incubation of L-leucine at 30° with Mg2+-activated leucine aminopeptidase in a solution that was enriched in oxygen-18 containing water for 24 h at pH 7.0 resulted in a substantial increase in the oxygen-18 content of the L-leucine as compared with blanks. When L-leucine, enriched with oxygen-18, was treated similarly with leucine aminopeptidase, a significant decrease in the oxygen-18 content occurred as compared with blank solutions and a heat-denatured leucine aminopeptidase control.
Evidence for an Essential Carboxyl Group in Glycogen Phosphorylase
Authors: Avramovic-Zikic O. , Breidenbach W. C. , Madsen N. B.
Published: December 2009

Phosphorylase b is rapidly inactivated by 5-diazo-1H-tetrazole or by 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate in the presence of glycine ethyl ester. In either case, incorporation of approximately 1 mol of reagent per mole of monomer results in complete inactivation. Consideration of the conditions for inactivation and the known specificity of the carbodiimide reagent suggests that modification of a carboxyl group may be causing the inactivation.
Differential Effect of Puromycin on Triacylglycerol and Phosphatidylcholine Synthesis in Rat Mecosal Microsomes
Authors: O'Doherty P. J. A. , Kuksis A.
Published: December 2009

The transferases involved in triacylglycerol and phosphatidylcholine synthesis were studied in mucosal microsomes of normal and puromycin-treated rats. The mono- and diacylglycerol acyltransferases showed similar activity in both normal and puromycin-treated microsomes. Cholinephosphotransferase was inhibited 55–60% and lysophos-phatidylcholine acyltransferase was inhibited 35–40% in mucosal microsomes from puromycin-treated rats. These results demonstrate that puromycin inhibits cholinephos-photransferase and lysophosphatidylcholine acyltransferase without interfering with the acyltransferases involved in triacylglycerol synthesis. Since the transferases involved in phosphatidylcholine synthesis are largely located in the rough endoplasmic reticulum, it is suggested that the inhibition of these enzymes is due to the selective disassembly of the rough endoplasmic reticulum by puromycin as demonstrated elsewhere.
Changes in the Adenyl Cyclase System of Skeletal Muscle of Cold-Acclimated Rats
Authors: Muirhead Michelle , Himms-Hagen Jean
Published: December 2009

No change could be detected in the adenyl cylase system (basal activity, noradrenaline-stimulated activity, adrenaline-stimulated activity, fluoride-stimulated activity) of skeletal muscle of cold-acclimated rats. It is concluded that the enhancement of metabolic response to noradrenaline during cold-acclimation, which occurs principally in skeletal muscle, can not be attributed to an alteration in this component of the receptor system for noradrenaline.An increase in the activity of the catecholamine-stimulated and fluoride-stimulated adenyl cyclase of skeletal muscle occurs during the 1st week of exposure to cold and has disappeared by the time the rats are fully acclimated. This increase coincides with the period of shivering thermogenesis rather than with the development of nonshivering thermogenesis; it may be related to the intense and repeated stimulation of plasma membrane associated with shivering.
Studies on Corticosterone–Receptor Complexes from Mouse Placenta
Authors: Wong Ming D. , Burton A. F.
Published: December 2009

The in vitro binding of radioactive steroids to components of mouse placental nuclei and cytoplasm was investigated using Sephadex or charcoal to remove unbound steroid. Specificity was indicated in competition experiments using excess unlabelled competing steroids. Only the active glucocorticoids formed complexes that could be isolated from the nucleus. The binding properties of the cytoplasmic steroid–receptor complex were studied. From the time course of binding the complex was shown to be more stable at 0° than at 37°, and the distribution of receptors in the cytosol appeared to be homogeneous. The complex was labile to heat and to proteolytic digestion but did not appear to be affected by nucleases or sulfhydryl reagents. Kinetic analysis revealed the presence of high affinity specific binding sites with a dissociation constant of 17.5 nM and a receptor site concentration of 0.26 pmol/mg protein. The corticosterone isolated from nuclear complexes and dexamethasone from cytoplasmic complexes were identified by chromatography and by cocrystallization as the unchanged steroid in each case.
Inhibition of Lipogenesis by Cyclic AMP Without Altered Lipolysis
Authors: Murthy Veeraraghavan K. , Steiner George
Published: December 2009

The microsome-free supernatants of rat interscapular brown fat synthesized fatty acid from acetate in the presence of a NADPH-generating system. Addition of cyclic AMP to the incubation system led to a concentration-dependent inhibition of fatty acid synthesis. The presence of cyclic AMP did not alter the concentration of long chain fatty acyl-CoA, free fatty acid, or triglycerides in the incubation mixture. These data suggest that cyclic AMP inhibited fatty acid synthesis through some mechanism other than an action on lipolysis.
The Resonance Raman Spectrum of the Metalloprotein Ovotransferrin
Authors: Carey Paul R. , Young N. Martin
Published: December 2009

Using ovotransferrin it is shown that amino-acid side chains forming the metal binding site in coloured metalloproteins can be identified and monitored by resonance Raman spectroscopy. All major features above 800 cm−1 in the resonance Raman spectrum of iron–bicarbonate ovotransferrin could be assigned to histidine or tyrosine vibrational modes, by comparison with Raman and infrared data for these amino acids and related compounds. Comparison of the features due to ring modes of imidazole ligated to Fe3+ in ovotransferrin with those of aqueous histidine at pH 8.0 suggests that rearrangement of the ring π electrons takes place upon ligation. Although iron–bicarbonate and iron–oxalate ovotransferrin have similar visible absorption spectra, differences in their resonance Raman spectra are clearly distinguishable, but no evidence was found for the direct binding of bicarbonate or oxalate to the iron. Differences between the geometries of the two sites at which iron is bound in iron–bicarbonate ovotransferrin appeared to be small compared with the perturbation caused to these sites by replacing bicarbonate with oxalate.
The Interaction of Ethidium Bromide with Synthetic Polydeoxyribonucleic Acids. Effect of Base Composition and Sequence on the Induced Circular Dichroism Spectra
Authors: Williams R. E. , Seligy V. L.
Published: December 2009

The interaction of the intercalating dye ethidium bromide with DNA and several synthetic polydeoxyribonucleic acids has been studied by means of circular dichroic spectra. All systems studied exhibited two strong dye-associated transitions at approximately 310 and 340 nm that were independent of polynucleotide base composition and sequence. Increasing dye – nucleotide phosphate ratios caused alterations in the spectra below 300 nm, suggesting that this dye may also cause changes in the polynucleotide conformations. Some slight specificity for alternating sequences of purines and pyrimidines, such as poly(dA-dT)/(dA-dT) was also noted.
Stability of Secretory Granules Containing Growth Hormone and Prolactin from Bovine Anterior Pituitary Gland
Authors: Lemay André , Labrie Fernand , Drouin Denis
Published: December 2009

Bovine adenohypophyseal secretory granules were purified by a technique giving a high yield of large granules containing 70–90% of prolactin (PRL) and 10–20% of growth hormone (GH). Purity of the preparation was checked by electron microscopy and enzymatic markers. Stability of the isolated secretory granules was studied by measurement of the optical density of the granule suspensions and by measurement of GH and PRL release by disc gel electrophoresis at pH 8.9. At 0 °C, granules suspended in an hypotonic medium are stable at pH 7.4. At room temperature, they are particularly stable at acidic pH but are almost completely solubilized at a pH ranging from 7.0 to 10.0, a half-maximal effect on PRL release being observed at pH 7.5. Lysolecithin has a marked solubilizing effect at a pH of 3.0–6.0 whereas 0.15% sodium dodecyl sulfate does not affect granule stability at a pH below 5. ATP (0.5 mM) inhibits GH and PRL release to, respectively, 65 and 27% of the control rates during a 60 min incubation at 37 °C and pH 7.4. A concentration of 2 mM Ca2+ has a marked inhibitory effect on both GH and PRL release whereas 2 mM Mg2+ inhibits PRL release but does not affect the release of GH. EGTA increases PRL release by 30% but does not significantly affect GH release. As evidenced by polyacrylamide gel electrophoresis, no degradation of GH or PRL occurred during a 1 h incubation of secretory granules at 37 °C and pH 7.4. These data suggest a role of Ca2+- and Mg2+-ATPase activities on the stability and possibly the formation of GH- and PRL-containing secretory granules.
Conversion of Pig Prothrombin into Thrombin: Amino Acid Composition of the Active Enzyme
Authors: Rossi Antonio , Giglio José Roberto
Published: December 2009

Preparation and activation of partially purified pig prothrombin are described. The enzyme, after column chromatography in Amberlite IRC-50, showed alanine as the N-terminal residue and an [Formula: see text] of 1.50 at pH 7.0 and 280 nm.The amino acid composition of pig thrombin is reported and compared with that of bovine origin. Pig thrombin showed 1.0 mol of isoleucine to 0.6 mol of threonine as N-terminals.The effects of storage of pig prothrombin are discussed.
Glycosylation of β-Lipoprotein and the Possible Role of Choline Derivatives
Authors: Delahunty Thomas , Mookerjea Sailen
Published: December 2009

(1) The incorporation of [1-14C]glucosamine and UDP-N-acetyl-[1-14C]glucosamine into β-lipoprotein was studied with rat liver slices and subcellular particles, respectively. β-Lipoprotein released into the medium was isolated by a modified heparin precipitation technique. The lecithin–protein ratios of heparin precipitable β-lipoprotein from the medium of liver microsomes and of serum were found to be very similar. With microsomes a substantial incorporation was obtained which reached a maximum in 12 min and 27 °C. The enzymatic attachment of the amino sugar to the apoprotein was further indicated when the incorporation was correlated with the quantity of microsomal protein present.(2) As shown previously with other protein acceptors, the addition of choline derivatives caused a considerable stimulation in incorporation of the label for slices, microsomes, and purified rough microsomes. The effect of CDP-choline on glycosylation was not as great when a Golgi-rich membrane fraction was used, suggesting that the major stimulation occurred at the sites of lipoprotein synthesis rather than secretion.(3) The release of prelabelled β-lipoprotein from liver slices was not enhanced by the addition of phosphorylcholine or excess glucosamine to the medium, suggesting that increased carbohydrate coupling does not stimulate secretion.
The Amino Acid Sequence of Streptomyces griseus Trypsin. II The Tryptic Peptides
Authors: Jurášek L. , Smillie L. B.
Published: December 2009

Streptomyces griseus trypsin (S.G.T.) isolated from pronase was reduced, aminoethylated, and digested with trypsin. The soluble peptides were recovered and the insoluble residue redigested with chymotrypsin. Following recovery of the soluble fraction, the insoluble portion was in turn digested with α-lytic protease of Myxobacter 495. The three groups of soluble peptides were separately subjected to ion-exchange chromatography on the Technicon peptide analyzer and to final purification by high-voltage paper electrophoresis. Sequence analysis by the dansyl-Edman procedure provided unique sequences from the soluble tryptic peptides accounting for 65% of the S.G.T. molecule. Peptides obtained from the redigests of the insoluble residues accounted for an additional 20%.Tryptic digestion of dansylated S.G.T. yielded a unique α-NH2 dansylated peptide whose composition showed it to be the same as the NH2-terminal sequence previously postulated for this enzyme. The tryptic peptides isolated in this work have provided overlaps for many of the previously sequenced peptic peptides. Three continuous sequences of 49, 36, and 28 residues have been elucidated.Evidence has also been obtained that the NH2-terminal valine residue was, to a limited extent, aminoethylated during reaction of the reduced protein with ethylenimine.
Patterns of Incorporation of [3H]leucine and [32P]orthophosphate into Nonhistone Proteins of Rainbow Trout during Early Development
Authors: Trevithick J. R.
Published: December 2009

In rainbow trout eggs developing at 10 °C, the relative amounts of nonhistone proteins of the embryo nuclei change during the course of cleavage; the relative amounts of at least two of the proteins seem to increase whereas four decrease during this period. [4,5-3H]leucine is incorporated into all components that stain visibly with Coomassie blue from mid-blastula on, and into approximately seven components at early blastula. [32P]orthophosphate is incorporated into three of the components of the nonhistone proteins, which also stain intensely with Coomassie blue. The results support a role for the nonhistone proteins in the control of differentiation and are in sharp contrast to the lack of change in the relative concentrations of the histones following the late blastula stage.
Origine de l'Inosine dans les tRNA de Levure
Authors: Dugré M. , Cedergren R. J.
Published: December 2009

The origin of inosine in the yeast tRNA anticodon was studied using a mutant of Saccharomyces cerevisiaewhich was adenylosuccinate lyase deficient and thus needed adenine as a growth factor. The radioactivity associated with the inosine was measured in the tRNAs labelled by [8-14C]adenine in the presence or absence of hypoxanthine. In both conditions the ratio of the radioactivity found in the fractions IMP and AMP remained constant. The inosine is thus the result of a post-transcriptional modification of adenosine. The fact that the biosynthesis of inosine is the same in yeast and bacteria allows the deduction of certain aspects of the evolution of tRNA.[Traduit par le journal]
The Enzymatic Synthesis of L-Cysteine in Higher Plant Tissues
Authors: Ngo T. T. , Shargool P. D.
Published: December 2009

Two enzymes that are together involved in the biosynthesis of cysteine in higher plants, O-acetyl-L-serine sulfhydrylase and L-serine acetyltransferase, have been isolated from the germinating seeds of different species of rape (Brassica spp.), and some of their properties studied.It is shown that some of the properties of the acetyltransferase enzyme differ markedly from those reported for the enzyme isolated from kidney bean seedlings. No correlation could be found between the levels of O-acetyl-L-serine sulfhydrylase and thioglucoside levels in the seeds of the various species of rape studied. The occurrence of this enzyme in a number of other species of flowering plants is also shown.
Purine and Pyrimidine Pool Sizes and Purine Base Utilization in Human Lymphocytes and Cultured Lymphoblasts
Authors: Fields T. , Brox L.
Published: December 2009

Purine base metabolism and purine ribonucleotide interconversions were compared in three lines of cultured human lymphoblasts as well as in normal human peripheral lymphocytes which were isolated by the Ficoll–Hypaque centrifugation procedure. In all cases, conversion of the purine base to the ribonucleoside monophosphate appeared to be rate-limiting. The relative rates of ATP and GTP synthesis from hypoxanthine were observed to vary in the different cells examined. Purine and pyrimidine ribonucleotide pool sizes, as determined by high-pressure liquid chromatography, are presented for the cultured cell lines.
A Study of the Conformational Properties of Glucagon in the Presence of Glycols
Authors: Contaxis C. C. , Epand R. M.
Published: December 2009

Elimination of the polymerization of glucagon has been achieved in the presence of glycols. The monomeric form of glucagon, found to be exclusively present under these conditions, is studied for its structural response to changes in the hydrophobicity of the environment. A reversible folding of the α-helix from about 0 to 80% is found to occur and it is suggested to proceed in two sequentially formed helical segments. Changes in the other spectroscopic properties are interpreted as changes in the tertiary structure. Implications for the biological activity are discussed.
Terminal Cross-linking of DNA Catalyzed by an Enzyme System Containing DNA Ligase, DNA Polymerase, and Exonuclease of Bacteriophage T7
Authors: Sadowski Paul , McGeer Allison , Becker Andrew
Published: December 2009

An enzymatic activity that terminally cross-links T7 DNA has been detected in extracts from phage T7-infected Escherichia coli cells. This activity required the functional products of gene 1.3 (T7 DNA ligase), gene 5 (T7 DNA polymerase), and gene 6 (T7 exonuclease) as well as Mg2+ ions, the four deoxytriphosphates, and ATP. The cross-linking system could be reconstituted with purified ligase, polymerase, and exonuclease, and maximal activity required the presence of all three enzymes. The system was active on phage DNA which possessed unique termini but not on DNA with non-unique ends.
Sequence Determination of a Protein with N-Terminal Glutamic Acid after Modification of Carboxyl Groups with Carbodiimide as the Coupling Agent
Authors: Gilardeau C. , Chrétien M.
Published: December 2009

Glutamine and asparagine residues in proteins can be differentiated from glutamic and aspartic residues, during the Edman degradation, after modification of the carboxyl groups by glycine methyl ester in presence of a water-soluble carbodiimide. When applied to ovine and porcine beta-lipotropic hormones, which have a glutamic acid residue at the N-terminus, the carbodiimide blocks the N-terminus. However, the Edman degradation proceeds normally, if the phenylthiocarbamyl derivative is formed prior to the modification reaction with glycine. In this communication, radioactive glycine was used to modify the carboxyl groups.
Some Physical Parameters of Succinyl-Coenzyme A Synthetase of Escherichia coli
Authors: Krebs Anita , Bridger William A.
Published: December 2009

A physical study of succinyl-coenzyme A synthetase of Escherichia coli has been conducted. The extinction coefficient for the enzyme at 280 nm [Formula: see text] has been evaluated by two independent methods and found to be equal to 4.9 ± 0.2. Sedimentation equilibrium studies show that there is a marked dependence of the apparent molecular weight upon the concentration of the enzyme. At concentrations above 1 mg/ml, the enzyme exists predominantly as an α2β2 tetramer of overall molecular weight near 140 000; at lower concentrations, a significant fraction of the enzyme dissociates to an αβ dimer. The circular dichroism spectrum of the enzyme suggests a high proportion of random coil structure, with small contributions of α-helix and β-structure.
The Influence of Ligands on the Fluorescence of the Complex between 8-Anilino-1-naphthalene Sulfonate and Pyruvate Kinase of Neurospora
Authors: Kapoor M.
Published: December 2009

The fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) bound to pyruvate kinase (PK) was diminished by the allosteric effector fructose 1,6-diphosphate (FDP). One of the substrates, phosphoenolpyruvate (PEP), stabilized the binding of FDP to the enzyme. The fluorescence of ANS and its reduction by FDP was studied as a function of temperature and pH: at low temperatures (12–15 °C) the relative quantum yields of the bound ANS were high but virtually no binding of FDP occurred. The optimum temperature and pH for FDP binding was observed to lie in the range 22–26 °C and 7.0–8.0, respectively. On incubation of PK with ANS at 22 °C and 35 °C under conditions corresponding to those employed for fluorescence studies, no inactivation of the enzyme was encountered; incubation at 3–4 °C led to a loss of 85–90% of the activity in 60 min. PEP and FDP, but not ADP, protected the enzyme against ANS-induced inactivation. In contrast, inactivation experiments conducted with rabbit muscle PK showed that both PEP and ADP afforded protection but FDP was completely ineffective.
An Atypical Human Immunoglobulin G with Deletions in both Heavy and Light Chains. Studies of the Conformation and the In Vitro Recombination of the Isolated Subunits
Authors: Percy M. E. , Dorrington K. J.
Published: December 2009

Both the light and gamma (heavy) chains of IgG(Sac) contain extensive deletions in their variable regions. The deletion in the light chain is internal (residues 18–88), whereas the deletion in the heavy chain is amino-terminal (residues 1–102). The hypervariable region just preceding the beginning of the constant region in other heavy chains (residues 103–115) is amino-terminal in heavy chain(Sac). In 4 mM acetate, pH 5.4, heavy chain(Sac) is dimeric like normal gamma chains, whereas light chain(Sac) is monomeric. Isolated light and heavy chains of IgG(Sac) recombine in vitro with each other and also with the heavy and light chains from a typical human IgG1-K myeloma protein, but not in a fashion entirely typical of other human gamma and light chains. These studies support the concept that non-covalent forces between the variable regions of the light and heavy chains are important in the assembly of the immunoglobulin molecule; and in view of the weak interaction between the constant region of light chain and heavy chain observed previously, our data suggest that there are points of contact between the hypervariable region of the gamma chain (residues 103–115) and the variable region of the light chain.
The Isolation of Trypsin and Elastase from Moose Pancreas (Alces alces) by Affinity Chromatography on Lima-Bean Protease Inhibitor – Sepharose Resin
Authors: Lievaart Paul A. , Stevenson Kenneth J.
Published: December 2009

Chymotrypsin, trypsin, and elastase were isolated from pancreas glands of the moose (Alces alces) by acid extraction, (NH4)2SO4 fractionation, and affinity chromatography conducted sequentially on 4-phenylbutylamine (PBA) – Sepharose and lima-bean protease inhibitor (LBI) – Sepharose. The removal of chymotrypsin on PBA–Sepharose led to the coabsorption of trypsin and elastase on to LBI–Sepharose. The binding site of elastase was identified as the 'chymotrypsin-site' of LBI. Elastase and trypsin were each selectively desorbed from LBI–Sepharose. These proteases exhibited identical elution profiles during carboxymethylcellulose chromatography and had similar migration rates on polyacrylamide disc gel electrophoresis. Moose elastase contained some trypsin activity whereas the trypsin preparation was pure based on comparison of ionograms of peptides derived from the proteolytic digestion of glucagon by moose chymotrypsin, trypsin, elastase, bovine trypsin, and porcine elastase.
Studies of the Metabolism of Asialotransferrins: The Mechanism for the Hypercatabolism of Human Asialotransferrin in the Rabbit
Authors: Regoeczi E. , Hatton M. W. C.
Published: December 2009

The behavior in vivo of human asialotransferrin in rabbits was studied in such a manner as to permit a comparison of this catabolic process with the generalized hepatic clearance mechanism for asialoglycoproteins described by Ashwell and Morell.Progressive desialylation of human transferrin in the range 50–100% yielded transferrin molecules with circulation times inversely proportional to the loss of sialic acid. The shape of plasma protein-bound radioactivity curves indicated that treatment of transferrin with neuraminidase resulted in at least two types of asialo derivatives. The biological half-life of one of them was close to 60% of that of control transferrin and the other one disappeared from the plasma with a half-life of approximately 1.5–2.5 h. Oxidation of over 50% of the terminal galactosyl groups in human asialotransferrin prolonged the circulation time of asialotransferrin.Assays of tissue radioactivities following mixed injection of human control and asialotransferrins showed that the two proteins possessed the same affinity for lung, kidney, and spleen, but asialotransferrin was preferentially taken up by the liver.On the basis of these observations, it seems likely that the mechanism which has been claimed as a generalized pathway for the clearance of asialoglycoproteins is also responsible for the rapid elimination of heterologous human asialotransferrin in the rabbit.