bluuebunny/crossref_metadata_2025 huggingface dataset:
N-Acetylglucosaminyltransferase Activity in Liver, Serum, and Ovaries of Domestic Fowl
Authors: Gornall D. A. , Kuksis A. , Pinteric L. , Mookerjea Sailen
Published: December 2009

Homogenates of the liver, serum, and ovaries of the laying hen and the liver and serum of the immature pullet were examined for N-acetylglucosaminyltransferase activity. The activity in the liver homogenates was higher in the laying hen than in the pullet while their sera showed similar low levels of enzyme activity. The homogenate of the ovary from the laying hen was also found to contain N-acetylglucos-aminyltransferase. Golgi-rich and Golgi-depleted membrane fractions were prepared from the liver homogenates of both types of fowl and examined in the assay system. The Golgi-depleted membrane fractions from the livers of both laying hen and pullet had higher enzyme activities than the corresponding Golgi-rich fractions. The N-acetylglucosaminyltransferase activities in both cellular membrane fractions were higher in the laying hen than in the pullet. The results are compatible with the hypothesis that N-acetylglucosaminyltransferase is involved in synthesis of lipoproteins and/or glycoproteins by the liver and the ovary. In contrast to the rat, the hen enzyme did not show increased activity in the presence of added CDP-choline.
The Temperature Dependence of the Electron Paramagnetic Resonance Spectrum of Ferricytochrome c Solutions Between 4.2 °K and 77 °K
Authors: Mailer C. , Taylor C. P. S.
Published: December 2009

Electron paramagnetic resonance (E.P.R.) signals from tuna ferricytochrome c solutions were obtained in the temperature range 4.2–77 °K. The microwave power and temperature dependence of the spectra obtained fit the theory for a Kramer's doublet split by a magnetic field. The g-values obtained are 1.25, 2.24, and 3.05 in agreement with other workers (Salmeen, I., and Palmer, G.: J. Chem. Phys. 48, 2049 (1968)). The g = 3.05 line changes from 380 G wide at 20–40 °K with Gaussian shape to 700 G wide at 77 °K with Lorentzian shape. Analysis shows that the Gaussian shape can be explained by variation in the rhombic symmetry of the heme iron environment, a ± 6% spread in rhombic potential being required to give a 380 G line width. At higher temperatures the line appears to be determined by the electron spin-lattice relaxation time. Below 20 °K the E.P.R. absorption signal is saturated, and a large E.P.R. dispersion signal appears, due to fast passage effects, which can be used to give further information on electron spin relaxation times.
Metabolism of Amino Acids in Plants. I. Changes in the Soluble Amino Acid Fractions of Bushbean Seedlings (Phaseolus vulgaris L.) and the Development of Transaminase Activity
Authors: Forest J. C. , Wightman F.
Published: December 2009

The total nitrogen content of seedlings grown under light or dark conditions remained relatively constant during the first 2 weeks of growth, but there was considerable redistribution of nitrogen from cotyledons to the seedling tissues and appreciable changes occurred in the ratio between insoluble and soluble nitrogen in the different organs. Soluble nitrogen increased in etiolated seedlings to reach 55% of the total nitrogen at 14 days, while in light-grown seedlings the soluble nitrogen reached a peak on the 8th day and thereafter rapidly decreased to 25% of the total nitrogen by the end of 2 weeks. The quantitative composition of the soluble nitrogen fraction, with respect to free protein amino acids, was found to differ considerably, not only between seedlings at different stages of growth, but also between organs taken from seedlings at the same stage of growth under the two light conditions. Asparagine, serine, valine, alanine, and threonine were the amino acids that showed the greatest change in amounts in both types of seedlings. Of the six transaminases investigated, only aspartate, alanine, and asparagine aminotransferase systems showed high total activity. Valine aminotransferase had low activity, and the serine and threonine transaminases were not detected. Development of the different transaminase activities was quite similar in seedlings grown under light or dark conditions. No correlation was observed between the development of transaminase activities and the changes in concentrations of the corresponding free amino acids in these seedlings.
The Acylation of Ribonucleotides with Benzoic and Acetic Anhydrides in Aqueous Solutions
Authors: Cedergren R. J. , Larue Bernard , Laporte Pierre
Published: December 2009

Benzoic and acetic anhydrides react in aqueous solutions with monoribonucleotides to give exclusively O-acyl derivatives. The order of reactivity with benzoic anhydride is pG, pA, pU, and pC; pG being five times more reactive than pC (pN = nucleoside 5′-phosphate). The relative reactivity of the nucleotides may be a result of 2′-hydroxyl interaction with the base.
Kinetics of the Reaction of p-Chloromercuribenzoate with the Sulfhydryl Groups of Glutathione, 2-Mercaptoethanol, and Phosphorylase b
Authors: Hasinoff B. B. , Madsen N. B. , Avramovic-Zikic O.
Published: December 2009

The reaction of p-chloromercuribenzoate (PCMB) with the sulfhydryl groups of phosphorylase b and the model compounds glutathione and 2-mercaptoethanol was followed spectrophotometrically on a stopped flow apparatus. Model sulfhydryl compounds, glutathione and 2-mercaptoethanol, under identical conditions of pH (6.8), buffer, and temperature (30°) reacted at a rate only slightly higher (6 × 106 M−1 s−1) than the fastest-reacting sulfhydryl groups of phosphorylase b (3 × 106 M−1 s−1). The sulfhydryl groups which are concerned with structure–activity relationships react at rates which are four to five orders of magnitude less than these.An analysis of the pH dependence of the rate of reaction of PCMB with the fastest-reacting groups of phosphorylase b and with 2-mercaptoethanol indicated that the ionized sulfhydryl group is reacting at a much faster rate than is the unionized group. The reaction of PCMB with the ionized sulfhydryl group is so fast (~ 106M−1 s−1) that it is presumably diffusion controlled, while the reaction with the unionized group, although three orders of magnitude slower, is still much greater than with those sulfhydryl groups concerned with structure and activity. Thus the reactivity of sulfhydryl groups of protein molecules is determined both by their pKa values and their accessibility to PCMB.
Studies on Spermatogenesis in Rats. II. Evidence that Carnitine Acetyltransferase is a Marker Enzyme for the Investigation of Germ Cell Differentiation
Authors: Vernon R. G. , Go V. L. W. , Fritz I. B.
Published: December 2009

Levels of carnitine acetyltransferase (CAT) in testes from neonatal rats were approximately 5% of those obtained in testes from adult rats. The changing activities measured during development indicated that maximal rates of increase were achieved at a time when mature primary spermatocytes were being generated. Spermatogonia had lowest CAT levels (0.16 unit/108 cells) while diplotene primary spermatocytes had highest CAT activities (6.1 units/108 cells). The apparent specific activities of other mitochondrial enzymes measured (glutamic dehydrogenase, carnitine palmitoyltransferase, and cytochrome oxidase) in testis did not increase at a time when CAT levels were elevated by over sevenfold.In adult hypophysectomized rats, CAT levels in testes decreased during the regression period, with most rapid rates of loss of activities at times corresponding to the diminution of spermatids and spermatocytes. The CAT activities in testes of fully regressed hypophysectomized rats were low, but were slightly higher than values obtained in testes from 7- and 14-day-old rats. The data were discussed in relation to the use of CAT levels as a biochemical indicator of the relative number of spermatogonia and spermatocytes in rat testes.
Aspartyl Transfer Ribonucleic Acid Synthetase from Bakers' Yeast
Authors: Diller Robert F. B. , Tener G. M.
Published: December 2009

An aspartyl-tRNA synthetase has been purified from the genetic standard type yeast Saccharomyces cerevisiae S288C. Lysis with toluene was followed by six column chromatographic purification steps in the presence of glycerol. The enzyme was purified 166-fold with 12% recovery relative to activity in the initial chromatographic fraction. Significant activation occurred during chromatography on the cation exchange resin, Bio-Rex 70. The purified enzyme was free of other aminoacyl-tRNA synthetase activities tested except a trace of that for glutamyl-tRNA and was essentially pure by acrylamide gel electrophoresis.
Metabolites of Verticillium dahliae: The Isolation and Structure Elucidation of Some Disaccharides and a Trisaccharide
Authors: Choy Y. M. , Unrau A. M.
Published: December 2009

Analysis of the concentrated culture filtrates by paper chromatography showed the presence of a series of oligosaccharides. Partial resolution of the saccharides was accomplished by charcoal–celite column and paper chromatography. 6-O-β-D-Fructofuranosyl-D-glucose and 4-O-β-D-fructofuranosyl-D-glucose were identified by methylation and hydrolysis followed by g.l.c. analysis of the component O-methyl sugars. Similarly, the occurrence of 2-O-β-D-fructofuranosyl-D-fructofuranose, a unique difructose, was demonstrated and evidence is presented for the presence of minor amounts of 1-O-β-D-fructofuranosyl-D-fructose and 6-O-β-D-fructofuranosyl-D-fructose. Of the series of higher oligomers, the structure of one trisaccharide (a difructosylglucose) was determined.
Studies on the Biosynthesis of Mitomycin C by Streptomyces verticillatus
Authors: Bezanson G. S. , Vining L. C.
Published: December 2009

Of a series of 14C-labeled substrates tested as precursors of mitomycin C, D-glucose was the most effective. L-Methionine-methyl-14C specifically labeled the methoxyl but not the C-methyl substituent; several precursors which labeled the carbamyl group were probably first metabolized to carbon dioxide. Other results suggest that the carbon skeleton of mitomycin is not assembled from an aromatic precursor derived by either the shikimate or polyketide pathways, and is not a disguised sesquiterpene. The distribution of radioactivity in C-6 and the attached methyl group of mitomycin C labeled from glucose-1-14C, -2-14C, and -6-14C is consistent with derivation of the methylbenzoquinone moiety from a seven-carbon intermediate which, in turn, originates from glucose by reactions of the nonoxidative pentose phosphate pathway. An additional molecule of glucose may supply the six remaining carbon atoms of the mitomycin C skeleton.
Esterification of Linoleate by Rabbit Reticulocytes
Authors: Warrendorf Elizabeth M. , Rubinstein David
Published: December 2009

The rate of esterification of linoleate-1-14C by an erythrocyte suspension containing at least 80% reticulocytes was four times that of normocytes. The incorporation was primarily into phospholipids, with most of the radioactivity being found in lecithin. Incorporation of the linoleate-1-14C into neutral lipids was slight in the reticulocyte and not detectable in the normocyte. Glucose-U-14C and glycerol-1,3-14C were incorporated to the extent of 2–5% of the linoleate by the reticulocyte and not at all by the normocyte. On a cellular basis, the reticulocytes contained twice as much phospholipid as the normocytes, although they contained a significantly lower percent of lecithin and a higher percent of lysolecithin than the normocyte. Following incubation with linoleate-1-14C the reticulocytes were separated into membrane, mitochondrial, and microsomal fractions. Most of the esterified fatty acid was found in the membrane fraction, with a small, but consistent, incorporation occurring in the mitochondria. Incorporation into the mitochondria was insufficient to account for the increased esterification by reticulocytes. Elevation of the intracellular ATP level by 50% (the maximum obtainable with reticulocytes) or diminution of the ATP concentration to that of the normocytes had no effect upon linoleate esterification by reticulocytes. It is concluded that esterification by reticulocytes, although more active than normocytes, follows the same pathway and that the subcellular fractions have almost completely lost the ability to renew their lipids at this stage of maturation.
Cholesterol Metabolism and Vitamin B6. III. The Stimulation of Hepatic Cholesterogenesis in the Vitamin B6-Deficient Rat
Authors: Hinse C. M. , Lupien P. J.
Published: December 2009

The rate of incorporation of labeled acetate into cholesterol by liver slices of pyridoxine-deficient rats was found to be three times that of control rats; with liver homogenates the difference between the two groups was even greater. Using the CO2 trapping technique, a 30% decrease in the hepatic acetate pool size was observed in pyridoxine-deficient rats and a 20% increase in pair-fed rats. Activity of the Krebs cycle was decreased by a third in the pair-fed rats.
The In Vivo Effect of Adrenocorticotropin on the Biosynthesis of Flavin Nucleotides in Rat Liver and Kidney
Authors: Fazekas A. G. , Sandor T.
Published: December 2009

In the livers and kidneys of rats treated with adrenocorticotropin, the biosynthesis of flavin mononucleotide from 2-14C-riboflavine was found to increase significantly as compared to control values. The formation of flavin–adenine dinucleotide and tissular uptake of riboflavine were not influenced by the hormone.
Cholesterol Metabolism and Vitamin B6. IV. Synthesis of Cholic Acid Conjugates in Vitamin B6-Deficient Rats
Authors: Avery M. D. , Lupien P. J.
Published: December 2009

The in vitro conjugation of labelled free cholic acid with glycine and taurine in the livers of vitamin B6-deficient and pair-fed rats has been investigated. It appears from these results that vitamin B6-deficiency stimulates this conjugation, and some evidence is given which suggests that vitamin B6 may be responsible for the lower capacity of conjugation observed in the liver of the pair-fed rat.
Some Properties of the Succinate Dehydrogenase of Escherichia coii
Authors: Kim I. C. , Bragg P. D.
Published: December 2009

Succinate dehydrogenase (SDH) was found in a cell envelope fraction from E. coli. If the enzyme preparation was made in 0.01 M phosphate buffer, pH 7.5, with or without 0.025 M succinate, or in 0.01 M Tris buffer, the activity of the enzyme could be markedly increased by heating the enzyme at 38° for 15 min with 15 mM succinate. Activation did not occur at 0°. If the enzyme preparation was made in Tris buffer containing succinate the enzyme was found to be fully activated. SDH was obtained in a low molecular weight form by extracting an acetone powder of whole cells with buffer. This enzyme had a molecular weight of about 100 000 and could be purified free from cytochrome.
Electron Transport and Coupled Energy Generation in Pseudomonas saccharophila
Authors: Ishaque M. , Donawa A. , Aleem M. I. H.
Published: December 2009

The respiratory chain system of heterotrophically grown Pseudomonas saccharophila contained cytochromes of the b, c, a, and o types and also the NADH and succinate oxidase enzyme systems. Cell-free extracts catalyzed phosphorylation coupled to the oxidation of NADH, succinate, and ascorbate (plus cytochrome c). The P/O ratios were in the range of 1.00 for generated NADH, 0.29 for added NADH, 0.50 for succinate, and 0.25 for ascorbate (plus cytochrome c).The oxidative phosphorylation was uncoupled by 2,4-dinitrophenol, 2,6-dibromophenol, pentachlorophenol, m-chlorocarbonyl cyanide phenylhydrazone, and dicumarol without any inhibition of oxygen consumption. Phosphorylation coupled to NADH oxidation was completely inhibited by the flavoprotein inhibitors such as rotenone, amytal, and atabrine; these inhibitors had no effect, however, on the ATP synthesis associated with succinate oxidation. Antimycin A or 2-n-nonyl-4-hydroxyquinoline-N-oxide as well as cyanide or azide at low concentrations completely inhibited the phosphate esterification coupled to the oxidation of NADH or succinate, but had little or no effect on the oxygen consumption. Relatively higher concentrations of oligomycin were required for a complete inhibition of the electron-transport-linked ATP formation.
Pattern of Incorporation of 32P into the Phospholipids of the Rat Tapeworm Hymenolepis diminuta
Authors: Webb R. A. , Mettrick D. F.
Published: December 2009

The incorporation of 32P-orthophosphate into the phospholipids of H. diminuta was followed for up to 4 h in vitro. The rates and patterns of incorporation of 32P into phosphatidc acid, phosphoinositide, phosphatidylserine, lecithin, lysolecithin, and phosphatidylethanolamine plus cardiolipin indicated that H. diminuta possesses mechanisms for the de novo synthesis of these phospholipids. The major pathway utilized by H. diminuta in fatty acid incorporation was via phosphatidic acid.
Comparison of (+)- and (−)-Marmesin as Intermediates in the Biosynthesis of Linear Furanoconmarins
Authors: Steck Warren , Brown Stewart A.
Published: December 2009

Marmesin has been identified by chromatography and ultraviolet absorption spectrum in extracts of Ruta graveolens, in confirmation of earlier conclusions from tracer experiments that it is an intermediate in furanocoumarin biosynthesis in this species. Trapping experiments with [2-14C]umbelliferone and (+)- or (−)-marmesin were inconclusive, but indicated that only the (+)-form is metabolically active in R. graveolens. This was confirmed in Ruta and also in Heracleum lanatum and Angelica archangelica by direct comparison feedings of [3-14C](+)- and (−)-marmesin. The (+)-isomer was an efficient furanocoumarin precursor, as before, while little if any utilization of (−)-marmesin could be demonstrated.
Seizures in Rats Associated with Divalent Cation Inhibition of Na+–K+-ATP'ase
Authors: Donaldson J. , St-Pierre T. , Minnich J. , Barbeau A.
Published: December 2009

In vitro determination of rat brain microsomal ATP'ase activity revealed specific inhibition of the Na+–K+-ATP'ase by cations in the order Zn2+ > Cu2+ > Fe2+ > Mn2+. Intraventricular injection of the same cations or of ouabain resulted in convulsions. Regional analysis of ATP'ase from brains of rats after convulsions showed inhibited Na+–K+-ATP'ase activity in hippocampus and hypothalamus. Hippocampus and hypothalamus were found to have the highest Na+–K+-ATP'ase activity in the rat brain.The potent inhibitors of Na+–K+-ATP'ase in vitro (ouabain, Zn2+, and Cu2+) were similarly effective in vivo (hippocampus and hypothalamus), while the inhibitors relatively ineffective in vitro (Fe2+ and Mn2+) were similarly of low potency in vivo. The potent inhibitors of Na+–K+-ATP'ase caused convulsions at low doses; the ineffective inhibitors caused convulsions only at very high doses.
A Modified Synthesis of α-N-carbobenzoxy-L-lysine and the preparation and analysis of mixtures of ε-N-methyllysines
Authors: Benoiton N. Leo , Demayo Ruth E. , Moore Graham J. , Coggins John R.
Published: December 2009

α-N-Carbobenzoxy-L-lysine has been obtained in 37% yield by carbobenzoxylation of L-lysine in 25% excess benzaldehyde followed by passage of the solution through a cupric carbonate – alumina column. Reaction of the product in aqueous solution with diazomethane, followed by deprotection, gave a mixture of ε-N-methyllysine (MML), ε-N,ε-N-dimethyllysine (DML), and ε-N,ε-N,ε-N-trimethyllysine suitable as a standard for chromatographic purposes. It has been demonstrated that when a mixture of MML and DML emerges as one peak when chromatographed with an amino acid analyzer, the relative concentrations of the two amino acids in the mixture can be established from the 570/440 nm absorbance ratio.
The Oxidation of Fatty-Acyl Derivatives by Mitochondria from Bovine Fetal and Calf Hearts
Authors: Brosnan J. T. , Fritz I. B.
Published: December 2009

Functional activity of the "external" carnitine palmitoyltransferase in intact mitochondria, prepared from hearts from various sources, was estimated by measuring respiration by mitochondria in the presence of palmitoyl-CoA plus or minus l-carnitine. When palmitoyl-CoA alone was substrate, respiration was not increased above the basal rate under all conditions examined. Addition of l-carnitine increased respiration, provided the ionic strength of the incubation medium was sufficiently high. In the presence of palmitoyl-CoA plus l-carnitine, the rate of respiration increased as the ionic strength was increased to physiological levels. In contrast, the increase in rate of oxygen consumption by heart mitochondria which followed the addition of palmitoyl-l-carnitine was relatively independent of the ionic strength of the medium.Mitochondrial fractions prepared from fetal bovine hearts were shown to possess "external" carnitine palmitoyltransferase activity, as judged by the ability of l-carnitine to stimulate respiration by mitochondria incubated with palmitoyl-CoA under various conditions. These data were discussed in relation to information available concerning the functions of different carnitine acyltransferases in mitochondria.
Differential Effects of α-Amanitin on RNA Polymerase Activity in Nuclei and Mitochondria
Authors: Menon I. A.
Published: December 2009

Concentrations of α-amanitin which strongly inhibit nuclear RNA polymerase activity in presence of Mn2+ and ammonium sulfate did not produce any significant inhibition of RNA polymerase activity in mitochondria from rat brain, spleen, liver, and kidney under identical conditions. Much higher concentrations of α-amanitin, however, inhibited the mitochondrial RNA polymerase. The effects of α-amanitin on the mitochondrial RNA polymerase activity in presence of Mg2+ were similar to those on nuclear enzyme under the same conditions; in both cases only relatively very high concentrations produced significant inhibition.
Studies on the Synthesis of Phosphoglycerides in Escherichia coli
Authors: Benns Georgina , Proulx P.
Published: December 2009

The incorporation of various labelled precursors into E. coli lipids was studied. In growing cells, incorporation of glycerol, doubly labelled with 14C and 3H, occurs without a change in isotope ratio. In cell homogenates incorporation of sn-glycero-3-phosphate-U-14C was greater in the polyglycerophosphatide fraction than in phosphatidylethanolamine and higher in the unacylated than in the acylated glycerol moiety of phosphatidylglycerol. This uneven distribution of label in phosphatidylglycerol was not affected by addition of unlabelled dihydroxyacetone phosphate and occurred to the same extent when glucose-3,4-14C was the precursor. Our results obtained in vivo indicate that glycerophosphate is acylated without prior oxidation. Furthermore, labelling patterns obtained in vitro suggest an extensive dilution by endogenous diacyl glycerol precursors not formed by acylation of dihydroxyacetone phosphate.
Rifamycin-Resistant Mutation in E. coli: Effect of Rifamycin on DNA-Dependent RNA Polymerase and on R17 Phage Growth
Authors: Igarashi S. J.
Published: December 2009

Rifamycin-resistant mutants in E. coli, K12, Hfr strain, were isolated without mutagenic treatment. The frequency of natural mutation of this type appeared to be approximately 10−9. Selection of resistant strains was effected by plating 109 cells on agar plates containing 50 μg of rifamycin per milliliter. A total of 11 strains resistant to rifamycin thus isolated remained Hfr and, in the presence of rifamycin, permitted the growth of the RNA-containing, male specific coliphage R17, which is sensitive to rifamycin in a wild-type E. coli. DNA-dependent RNA polymerase was prepared from these resistant mutants and examined for sensitivity toward rifamycin and other antibiotics. It was found that the RNA polymerase of the resistant strain is indeed resistant to rifamycin. However, both rifamycin-resistant and -sensitive enzymes are inhibited by the other three antibiotics (acridine orange, ethidium bromide, actinomycin D), which are known to interact with the template molecule.
On the Purification of DNA-Dependent RNA Polymerase from E. coli: Removal of an ATPase
Authors: Paetkau Verner , Coy Garry
Published: December 2009

DNA-dependent RNA polymerase has been purified from E. coli B grown to three-quarters log phase in minimal medium. The method of purification includes chromatography on Agarose 1.5 M, and QAE-Sephadex. The second of these steps resolves the RNA polymerase activity into two fractions. One of these has the composition (ββ′σα2). The other contains the "core" subunits β, β′, and α, and another protein whose molecular weight is 68 000 in sodium dodecyl sulfate. This latter protein can be separated from the core subunits, and is shown to have ATPase activity. The possible relationship of this protein to ones of similar molecular weight, found in RNA polymerase preparations of other authors, is discussed.
DNA-Dependent RNA Polymerase from Tetrahymena pyriformis
Authors: Kurtz Sonia , Pearlman Ronald E.
Published: December 2009

DNA-dependent RNA polymerase has been isolated from sonicated nuclei of the ciliate protozoan Tetrahymena pyriformis. The enzyme has been further purified by column chromatography on DEAE-Sephadex and is approximately 300-fold purified relative to total cellular protein.
Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties
Authors: Howden R. L. , Lees H. , Suzuki Isamu
Published: December 2009

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.
Effect of Chloramphenicol on Enzyme Synthesis and Growth of Mammalian Cells
Authors: Fettes I. M. , Haldar D. , Freeman K. B.
Published: December 2009

D-threo-Chloramphenicol and its sulfamoyl analogue Tevenel, but not L-chloramphenicol, prevent the formation of a functional cytochrome c oxidase in L cells. The syntheses of several mitochondrial enzymes, malate dehydrogenase, succinate dehydrogenase, NADH dehydrogenase, ATPase, and cytochromes b, c, and c1, were not inhibited. Cycloheximide also inhibited the formation of cytochrome c oxidase. After removal of D-chloramphenicol or Tevenel, cytochrome oxidase activity returned at a rate equivalent to its normal synthesis. L cells continued to grow for up to five generations in the presence of Tevenel at 100 μg/ml but only for 1.5 to two generations with D-chloramphenicol at the same concentration. D-Chloramphenicol specifically inhibited the synthesis of insoluble mitochondrial proteins in ascites cells.
DDT-Induced Stimulation of Key Gluconeogenic Enzymes In Rat Kidney Cortex
Authors: Kacew S. , Singhal R. L. , Ling G. M.
Published: December 2009

Administration of technical DDT or o,p′-DDT produced marked increases in pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-diphosphatase, and glueose-6-phospfaatase activities in rat kidney cortex. Significant increases in these key gluconeogenic enzymes occurred at 2–3 days and maximal stimulation was seen 5–7 days after the beginning of o,p′-DDT treatment. This DDT isomer, when given to adrenalectomized rats, produced increases in renal enzymes similar to those observed in intact animals. Furthermore, since administration of triamcinolone to o,p′-DDT-treated rats failed to potentiate the action of this insecticide on various enzymes, evidence indicates that the stimulation of kidney cortex gluconeogenesis by DDT is not mediated through a release of glucocorticoid hormones from the adrenal cortex.
Estimations of Rates of Synthesis and Degradation of Several Rat Mammary Gland Enzymes from Changes in Enzyme Activities
Authors: Korsrud G. O. , Baldwin R. L.
Published: December 2009

Based upon rates of decrease in the activities of citrate cleavage enzyme (EC 4.1.3.7), malic enzyme (EC 1.1.1.40), fatty acid synthetase, UDPglucose pyrophosphorylase (EC 2.7.7.9), UDPglucose 4-epimerase (EC 5.1.3.3), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in rat mammary glands after adrenalectomy–ovariectomy on the 11th day of lactation, the half-lives of the enzymes were estimated to be 28, 31, 28, 50, 20, and 24 h, respectively. The half-life estimates for UDPglucose pyrophosphorylase and glucose-6-phosphate dehydrogenase compared favorably with previous estimates of 35 and 20 h, respectively, obtained in rats 5 days postpartum utilizing specific immunological techniques. In a second experiment, increases in the activities of enzymes in adrenalectomized, lactating rats after initiation of cortisol therapy were investigated. Rats were adrenalectomized on the 5th day of lactation and cortisol therapy was started 5 days later. The estimated half-lives for citrate cleavage enzyme, malic enzyme, fatty acid synthetase, UDPglucose pyrophosphorylase, UDPglucose 4-epimerase, the A protein of the lactose synthetase complex, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and hexokinase (EC 2.7.1.1) were, respectively, 84, 60, 92, 76, 170, 102, 79, 88, and 81 h.
Influence of Anionic Surfactants and Ionic Strength on α-Coymotrypsin-Catalyzed Reactions
Authors: Gaudin J. E. , Viswanatha T.
Published: December 2009

The effect of sodium dodecyl sulfate and sodium decylbenzene sulfonate on the chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tyrosine ethyl ester was investigated. Pretreatment of the enzyme with sodium dodecyl sulfate produces inactivation which is dependent on the ratio of the surfactant to protein. Total loss of activity is achieved at a surfactant to protein (w/w) ratio of 15. Addition of chymotrypsin to a mixture of detergent and substrate does not affect the activity of the enzyme at low concentrations of sodium dodecyl sulfate. An enhancement in the activity is noted at higher surfactant concentrations, presumably due to the increase in the ionic strength of the reaction medium. The reaction of sodium decylbenzene sulfonate with chymotrypsin results in the formation of a series of complexes in an 'all or none' manner. Inactivation of the enzyme is noted initially at a surfactant to protein ratio of 1.0 and subsequently at ratios higher than 10. However, considerable activity is retained at surfactant to protein ratios of 3:5. The hydrolysis of N-acetyl-L-tyrosine ethyl ester is considerably influenced by the ionic strength of the medium. Increases in the ionic strength cause an enhancement in Vmax without affecting the affinity of the enzyme for the substrate.
Isolation and Characterization of Glycogen from Streptococcus salivarius
Authors: Khandelwal Ramji L. , Spearman Terry N. , Hamilton Ian R.
Published: December 2009

Highly purified glycogen was isolated and characterized from the oral microbe Streptococcus salivarius. The wavelength for the maximum absorption of the iodine complex, the specific optical rotation, along with the values for the average chain length, exterior chain length, and interior chain length, were within the range reported for other microbial and animal glycogens.
Fatty Acid Oxidation in Crithidia fasciculata
Authors: Vakirtzi-Lemonias C. , Karahalios C. C. , Levis G. M.
Published: December 2009

Cultures of the kinetoplastid Crithidia fasciculata at their logarithmic phase of growth oxidize [1-14C]palmitate to α-hydroxy fatty acids, CO2, and water-soluble products. 2-Hydroxy[1-14C]stearate is also decarboxylated. The decarboxylation of [1-14C]palmitate by cells and homogenate decreases in the presence of nonradioactive L-2-hydroxypalmitate, and to a lesser extent in the presence of the D- form. [2-14C]stearate, compared with [1-14C]palmitate, is oxidized to a greater extent into water-soluble products and this process is not affected by nonradioactive DL-2-hydroxystearate.Radioactive peaks corresponding to the mass peaks of odd-chain and α-hydroxy fatty acids have been detected by gas–liquid chromatography after incubation of whole cells or cell fractions with [9,10-3H2]palmitate or stearate.The results suggest the operation of the α-oxidation system for degradation of fatty acids to be via the α-hydroxy intermediate.
Membrane-Bound γ-Glutamyl Transpeptidase. Evidence that it is a Component of the 'Amino Acid Site' of Certain Neutral Amino Acid Transport Systems
Authors: Bodnaryk Robert P.
Published: December 2009

A membrane-bound γ-glutamyl transpeptidase is likely to be a key component of the 'amino acid site' of certain neutral amino acid transport systems. The hypothesis is in keeping with the Orlowski–Meister concept of a γ-glutamyl cycle for some forms of amino acid transport, and it is supported experimentally by a demonstration that the specificity of a γ-glutamyl transpeptidase (described herein) is virtually the same as the affinity of an amino acid site (described by Hajjar and Curran) for phenylalanine and 16 of its variously substituted analogues.Thus, both transpeptidase and transport site have a greater specificity for phenylalanine analogues having electron-withdrawing groups as substituents in the benzene ring than phenylalanine itself, and reduced specificity for analogues with electron-releasing substituents in the ring. The order of specificity according to ring substitution was −NO2 > −Cl > −F > −H > −CH3 > −OH > −NH2 for both systems. Again, the free α-amino group of phenylalanine plays a key role in the binding of the amino acid to the site, and it is also essential for the transpeptidase. Finally, the essential feature of the carboxyl group for binding of a neutral amino acid by the transport site is the −C = O group and this group is essential for maintaining the γ-glutamyl acceptor properties of an acceptor.
Studies on Plasminogen. X. Isolation of Plasminogen by Affinity Chromatography Using Sepharose–Butesin
Authors: Zolton Raymond P. , Mertz Edwin T.
Published: December 2009

Highly purified sheep, bovine, and human plasminogen can be prepared by affinity chromatography of the plasma or plasma euglobulin fraction on a column of Sepharose–Butesin (Sepharose–NH∙C6H4∙COOC4-H9). Since plasma is not bound, the column can also be used to monitor the conversion of plasminogen to plasmin.
The Partial Purification of Two β-N-Acetyl-D-hexosaminidases from Porcine Kidney
Authors: Wetmore Stephen J. , Verpoorte Jacob A.
Published: December 2009

Two distinct fractions showing both β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) and β-N-acetyl-D-galactosaminidase activity were isolated and purified from pig kidney. These preparations, which were designated A and B, were not stable during gel chromatography or prolonged dialysis. Final purifications of 600-fold for enzyme A and 440-fold for enzyme B were obtained.Gel electrophoresis and ultracentrifugation studies indicated heterogeneity in both preparations. The amino acid compositions of both preparations were very similar. Ultracentrifugation studies suggested the formation of subunits in the presence of 5 M guanidine–HCl and 1 mM dithiothreitol.A study of the enzymatic properties also showed great similarities between the two enzyme forms. Both enzymes had identical Michaelis–Menten constants of 1.88 mM for p-nitrophenyl-β-N-acetyl-D-glucosaminide and 0.38 mM for p-nitrophenyl-β-N-acetyl-D-galactosaminide. Although bovine serum albumin enhanced the activity of the enzymes it did not change the Km values. The pH-rate profiles of both enzymes with the substrate p-nitrophenyl-β-N-acetyl-D-glucosaminide showed two peaks. When p-nitrophenyl-β-N-acetyl-D-galactosaminide was used as substrate, only one peak was observed in the pH–rate profiles. However, in this case a distinct shoulder could be detected in these peaks. Heating at 50° destroyed the activities of both forms of the enzyme rapidly, but addition of bovine serum albumin protected against heat inactivation.
The Levels of Nucleotide-Sugar: Glycoprotein Sialyl- and N-Acetyl-glucosaminyltransferases in Normal and Pathological Human Sera
Authors: Mookerjea Sailen , Michaels M. Alex , Hudgin Roger L. , Moscarello Mario A. , Chow Annie , Schachter Harry
Published: December 2009

Enzymes which transfer N-acetylneuraminic acid and N-acetylglucosamine from their respective nucleotide-sugars to exogenously added glycoprotein acceptors are present in human sera. The levels of these enzymes have been determined in various pathological sera. Sialyltransferase and N-acetylglucosaminyltransferase levels were increased in a group of patients with various liver diseases. N-Acetylglucosaminyltransferase levels were also increased in a group of patients with a variety of infections. The data support the conclusion that these serum enzymes are derived at least in part from the liver.
Purification and Properties of Hog-Kidney 3,4-Dihydroxyphenylalanine Decarboxylase
Authors: Lancaster G. A. , Sourkes T. L.
Published: December 2009

A procedure for the purification of 3,4-dihydroxyphenylaianine decarboxylase from hog kidney is described. Enzyme fractions with 190-fold greater specific activity than in the supernatant portion of kidney cortex have been obtained. Such fractions yield a single protein peak by disc gel electrophoresis and by continuous electrophoresis in acrylamide gel. The purified enzyme is partially resolved with respect to its coenzyme pyridoxal phosphate. It appears to consist of two polypeptide chains of unequal size. The purified enzyme catalyzes the decarboxylation of dihydroxyphenylalanine, 5-hydroxytryptophan, erythro-dihydroxyphenylserine, m-tyrosine, and o-tyrosine, but not of p-tyrosine and threo-dihydroxyphenylserine.
Decrease of Insulin Immunoreactivity during Storage in the Presence of Aldoses
Authors: Mitsuhashi O. , Schucher R. , Kalant N.
Published: December 2009

Insulin stored in the presence of certain aldoses gradually lost its immunoreactivity; dilution, so that the aldose concentration fell below 5 mg/ml, and further storage resulted in gradual recovery of immunoreactivity. The loss of reactivity was not associated with a decrease in biological activity in the fat pad assay.
The Harbor Seal Phoca vitulina concolor De Kay: Comparative Details of Fatty Acids in Lung and Heart Phospholipids and Triglycerides
Authors: Ackman R. G. , Hooper S. N. , Hingley J.
Published: December 2009

The fatty acids of phospholipids and of triglycerides from the lungs and heart of the harbor seal Phoca vitulina concolor De Kay show distinctive differences from the depot (blubber) fat. Primarily, this is due to greatly elevated levels of 20:4ω6 in the phospholipids. Although these have about the same total C20 and C22 polyethylenic acids with five and six double bonds as the depot fat, they occur in different proportions emphasizing 20:5ω3 rather than 22:6ω3. The organ triglycerides resemble the phospholipids in saturated fatty acid composition and the depot fats in monoethylenic fatty acid composition, but have remarkably low levels of 20:5ω3, 22:5ω3, and 22:6ω3 compared to the depot fat. Comparisons are made with phospholipids from the lungs of the leatherback turtle Dermochelys coriacea coriacea Linnaeus but the novel trans-6-hexadecenoic acid found in the turtle lipids could not be detected in the seal lipids.
Pancreatic-Like Ribonuclease Activity in the Ascites Fluid of the Novikoff and Ehrlich Tumors
Authors: Miron Michel , Morais Réjean
Published: December 2009

The ascitic fluid of Novikoff and Ehrlich tumors contains a pancreatic-like ribonuclease. The enzyme catalyzes the hydrolysis of natural RNA and that of the synthetic pyrimidine (poly U, poly C) but not (poly A, poly G) or slightly (poly I) purine polymers. The synthetic duplex molecules poly I: poly C and poly A: poly U are rather resistant to hydrolysis.
Studies on Chymotrypsin-P. II. Kinetics
Authors: Shaw M. C. , Viswanatha T.
Published: December 2009

A comparison of the kinetics of chymotrypsin-P catalyzed hydrolysis of N-acetyl-L-tyrosine ethyl ester and cinnamoylimidazole with those of the chymotrypsin Aα catalyzed reactions revealed the two enzymes to possess very similar catalytic properties. The deletion of the three C-terminal residues from the A-chain of chymotrypsin Aα, a feature characteristic of chymotrypsin-P, does not lead to significant changes in the function of the enzyme.
Steroid Biosynthesis in Gonadal Tissue from Patients with Testicular Feminization
Authors: Cardiff C. , Uden V. , Killinger D. W.
Published: December 2009

The biosynthesis of sulfate conjugates and unconjugated steroids has been compared in two siblings aged 19 and 21, and a 52-year-old patient with the complete form of testicular feminization. Incubation of homogenates of gonadal tissue with 14C-pregnenolone (P) and 3H-pregnenolone-sulfate (PS) revealed that the conversion of P to PS was 6.8% in the older subject, and less than 1% in the two younger subjects; and the conversion to dehydroepiandrosterone sulfate (DHAS) was 24.2% in the older subject, and 2.1 and 0.2% in the two younger subjects. The recovery of unmetabolized PS was 41.9% in the older subject, and 5.0 and 13.7% in the two younger subjects. The formation of testosterone from P was 4.2 and 2.2% in the two younger subjects, and 0.5% in the older subject, and the 3H/14C ratio indicated that the utilization of PS in testosterone biosynthesis was greater in the younger patients than in the older subject. Incubation of 14C-dehydroepiandro-sterone (D) and 3H-DHAS with testicular tissue from the 21-year-old subject confirmed the high testosterone production and steroid sulfate utilization in the younger subject.These findings raise the possibility that with age there may be a shift from the biosynthesis of biologically active unconjugated products to less biologically active sulfate conjugates in testicular tissue.
Studies on Acute Phase Proteins of Rat Serum. II. Determination of the Contents of α1-Acid Glycoprotein, α2-Macroglobulin, and Albumin in Serum from Rats Suffering from Induced Inflammation
Authors: Jamieson J. C. , Ashton F. E. , Friesen A. D. , Chou B.
Published: December 2009

A quantitative precipitin technique has been employed to determine the contents of α1-acid glycoprotein, α2-macroglobulin, and albumin in serum from control rats and rats suffering from induced inflammation for 5–96 h. There was an increase in the content of α1-acid glycoprotein and α2-macroglobulin in serum from experimental animals reaching a maximum at 48–72 h after administration of inflammatory agent indicating that both proteins are acute phase globulins. There was only a slight change in the content of albumin in serum from experimental animals when compared with controls. Studies involving incorporation of labelled precursors of glycoprotein biosynthesis into α1-acid glycoprotein and α2-macroglobulin indicated that the most likely explanation for the increase in α1-acid glycoprotein and α2-macroglobulin in serum from experimental animals was an increase in the rates of synthesis of the two proteins in question.
Inhibition of DNA Polymerase of E. coli by Proflavine
Authors: Kadohama N. , McCarter J. A.
Published: December 2009

Proflavine (3,6-diaminoacridine) inhibits the synthesis of polynucleotides in vitro by DNA polymerase of E. coli (EC 2.7.7.7). The extent of inhibition of the enzyme by proflavine was a linear function of the concentration of the dye bound to DNA up to the point of saturation of strong binding sites (1 mol proflavine for approximately five base pairs).Kinetic studies of DNA polymerase and its inhibition by proflavine were made by following the rate of loss of thymidine triphosphate. The integrated form of the Michaelis–Menten equation allowed the apparent Km to be calculated for the poly (d(A–T)) reaction = 3.6 (S.D. ± 0.6) × 10−5 for six determinations. Inhibition by proflavine was competitive with respect to polynucleotide and the apparent Ki was 7.4 × 10−7 M. The number of nucleotides involved per inhibitory dye molecule was eight, i.e. the number of base pairs involved was four.For non-rate-limiting concentrations of polynucleotide the apparent Km for deoxyribonucleoside triphosphates (one nucleotide) was 5.4 (S.D. ± 3.3) × 10−5 M (five determinations). Inhibition by proflavine was noncompetitive and the apparent Ki was 1.9 × 10−5 M.It is suggested that these findings are accounted for by the increased stability of the acridine–polynucleotide complex.
Studies on the Role of Sialic Acid in the Physical and Biological Properties of Erythropoietin
Authors: Lukowsky W. A. , Painter R. H.
Published: December 2009

Crude erythropoietin was subjected to isoelectric focusing over the pH ranges 3–10 and 3–5. All the biological activity was located between pH 3.5 and 4.0. The erythropoietin activity displayed heterogeneity, being divided into three or four peaks within this pH range. Asialoerythropoietin, prepared by acid or enzymic hydrolysis, focused in the pH gradient between pH 5.0 and 6.5 and displayed a similar degree of heterogeneity. Partially desialated erythropoietin focused over a range of intermediate pH values. The large shift in pI on desialation and the demonstration of intermediate partially desialated species suggest a high content of sialic acid in erythropoietin. All desialated species of erythropoietin having isoelectric points greater than 4.0 were without in vivo biological activity. Oxidation of asialoerythropoietin with galactose oxidase completely restored in vivo biological activity.
Substrates for Ribosomal Peptidyl Transferase: Synthesis of 3′-N-Aminoacyl and 5′-O-Nucleotidyl Analogues of Puromycin
Authors: Harris R. J. , Mercer J. F. B. , Skingle D. C. , Symons R. H.
Published: December 2009

The chemical synthesis, on a 10–30 μmol scale, of two series of puromycin analogues is described: the first is the type 3′-N-aminoacyl-puromycin aminonucleoside (e.g. 3′-N-glycyl-PANS or PANS-Gly) in which the O-methyl-L-tyrosyl residue of puromycin is replaced by various aminoacyl residues, and the second is the type NpPANS-Gly in which the 5′-hydroxyl of PANS-Gly is substituted with phosphate, 3′-AMP, 3′-CMP, 3′-GMP, or 3′-UMP. The 3′-N-aminoacyl-PANS derivatives were synthesized either by coupling N-protected amino acids via their N-hydroxysuccinimide esters to PANS in 70% aqueous pyridine or by direct coupling in ethanol or methanol of N-protected amino acids to PANS using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as condensing agent After removal of the protecting groups, reaction mixtures were purified by preparative thin-layer chromatography on silicic acid, paper chromatography, and paper electrophoresis to give the 3′-N-aminoacyl-PANS derivatives in yields of 40–80%.The 5′-O-nucleotidyl derivatives of PANS-Gly were synthesized by three methods: (i) by the coupling of fully acetylated 3′-mononucleotides to 3′-N-(Boc-glycyl)-PANS, (ii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranylcytidine or N2,O2′,O5′-tritetrahydropyranylguanosine to 5′-O-phosphoryl-(Boc-glycyl)-PANS, and (iii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranyl-3′-CMP or N2,O2′,O5′-tritetrahydropyranyl-3′-GMP to 3′-N-Boc-PANS or 3′-N-trifluoroacetyl-PANS followed by aminoacylation of the CpPANS or GpPANS produced with Boc-glycine and EEDQ. Coupling reactions were carried out using either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulfonyl chloride as condensing agents in anhydrous pyridine. After removal of protecting groups, reaction mixtures were purified by paper chromatography and paper electrophoresis to give the NpPANS-Gly derivatives in 3–30% yield.