bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms for activation and inhibition of carboxypeptidase A catalyzed hydrolyses of peptides and esters
Authors: Sebastian John F. , Lo Whe-Yong
Published: December 2009

3,3-Diphenylpropanoate (DPP) activates the carboxypeptidase A catalyzed hydrolysis of benzoylglycyl-L-phenylalanine (BzGly-L-Phe) (Ka = 2.1 × 10−3 M) and inhibits ester hydrolysis uncompetitively (Ki = 2.1 × 10−3 M). A common modifier binding site located adjacent to the peptide and ester substrate binding sites is proposed. The forms of the pathways proposed for activation and inhibition are remarkably similar.
Assembly of the sarcoplasmic reticulum. Synthesis of calsequestrin and the Ca2++Mg2+-adenosme triphosphatase on membrane-bound polyribosomes
Authors: Greenway Donald C. , MacLennan David H.
Published: December 2009

Membrane-bound and free polyribosomes were isolated from skeletal muscle of neonatal rats and messages were translated in a rabbit reticulocyte lysate treated with a Ca2+-dependent nuclease to reduce endogenous messenger translation. Newly synthesized calsequestrin and adenosine triphosphatase (ATPase) were isolated by antibody precipitation, followed by separation of the precipitates in SDS-polyacrylamide gels. Radioactivity in calsequestrin and the ATPase were counted in gel slices. Calsequestrin and the ATPase were both found to be synthesized on membrane-bound polyribosomes. Since calsequestrin is a glycoprotein, localized in Golgi regions in early stages of muscle cell differentiation, it is probable that its synthesis follows the pathway for synthesis of secreted proteins except that its destination is the luminal space of a cellular organelle. The disposition of the ATPase during synthesis is, as yet, unknown.
Purification of the heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase by affinity chromatography
Authors: Sharma Rajendra K. , Desai Rekha , Thompson Thomas R. , Wang Jerry H.
Published: December 2009

The recently discovered heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase (Sharma, R, K., Wirch, E. &Warg, J. H. (1978) J. Biol. Chem., in press) has been purified 238 214-fold from bovine brain extract using an affinity column of the modulator protein – Sepharose 4B conjugate. The purified sample appears to be homogeneous as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The protein band has a mobility corresponding to that of a polypeptide of molecular weight 68 000. Since the heat-stable inhibitor protein has a molecular weight of 70 000 under nondenaturing conditions, it suggests that it is a monomeric protein. The protein has no inhibitory activity toward the cAMP-dependent protein kinase or protein phosphatase. The purified sample has been tested for various enzyme activities which include ATPase, GTPase, cAMP phosphodiesterase, cGMP phosphodiesterase, 5′-nucleotidase, and protein kinase. None of these activities are exhibited by the purified sample.
The purification and properties of valine tRNAs of Drosophila melanogaster
Authors: Dunn Robert , Addison W. R. , Gillam I. C. , Tener G. M.
Published: December 2009

The valine transfer ribonucleic acids of Drosophila melanogaster have been purified by column chromatography on BD-cellulose, Sepharose 6B, and RPC-5. Three major species were analyzed for base composition and coding properties. [Formula: see text] binds strongly to ribosomes in the presence of GUA and to a lesser extent with GUU and GUG. [Formula: see text] binds strongly in the presence of GUG and very poorly if at all with the other three triplets whereas [Formula: see text], which contains inosine, binds strongly in the presence of GUU, GUC, and GUA and weakly with GUG.
The nuclear magnetic resonance determination of the conformation of saccharides bound in subsite D of lysozyme
Authors: Patt Steven L. , Baldo John H. , Boekelheide Kim , Weisz Gregg , Sykes Brian D.
Published: December 2009

The binding of the trisaccharide (2-acetamido-2-deoxy-D-muramic acid)-β(1→4)-(2-aceta-mido-2-deoxy-D-glucosyl)-β(1→4)-(2-acetamido-2-deoxy-D-muramic acid) to subsites B, C, and D in lysozyme has been studied by 1H nuclear magnetic resonance methods. In particular, the coupling constant between H1 and H2 of the reducing saccharide bound in subsite D has been determined. The coupling constant for the bound saccharide indicates that the dihedral angle between C1 and C2 for the reducing saccharide is not significantly changed upon binding to lysozyme. This result is discussed in terms of other evidence for the role of distortion of the saccharide bound in subsite D in the lysozyme-catalyzed hydrolysis of cell wall oligosaccharides.
Regulation of the diurnal cycle in activity of serotonin acetyltransferase in the chick pineal gland
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

When chick pineal glands were explanted into organ culture at midlight phase of a diurnal cycle of illumination and incubated in the dark, they developed marked increases in serotonin acetyltransferase (acetyl coA:arylamine N-acetyltransferase; EC 2.3.1.5) activity. Either this increase in activity was inhibited or its onset was retarded in glands incubated under constant illumination.Supplements of theophylline, isobutylmethylxanthine, quinidine, and compound Ro 20-1724 (4-(3-butoxyl-4-methoxybenzyl)-2-imidazolidinone) elicited very marked increases in serotonin acetyltransferase activity in glands cultured in the dark. Levels of activity attained after 6 h in culture approached or exceeded the maximum levels attained at middark phase of the diurnal cycle in vivo. Effects of theophylline and compound Ro 20-1724 were additive.Supplements of dibutryl cAMP had little or no effect upon levels of serotonin acetyltransferase activity when tested alone or in combination with theophylline but further enhanced the increase in the level of enzyme activity elicited by Ro 20-1724. Adenosine and cAMP had little or no effect upon levels of serotonin acetyltransferase activity.It is concluded that levels of serotonin acetyltransferase activity in the chick pineal gland are regulated by a repressive, negative-control mechanism, which probably involves a membranous adenosine receptor.
The isolation and identification of 2,4-diketones present in human and animal tissues and in urine
Authors: Douglas D. E. , Francis L. E. , Melville K. I.
Published: December 2009

The chemical isolation of a 2,4-diketone fraction from hydrolysates of various mammalian tissues has been accomplished by the use of a modified Girard T procedure. This fraction, which constitutes a new lipid class, has been resolved by gas chromatography into a number of homologous 2,4-diketones, ranging in chain length from C13 to C25. After separation by preparative gas chromatography, the following compounds have been unequivocally identified: 2,4-heptadecanedione, 2,4-nonadecanedione, 2,4-heneicosanedione, 2,4-docosanedione, and Δ12-2,4-heneicosenedione. The 2,4-diketones appear to exist in tissues in the free state or in labile combination. They are present also in human urine.
Contrôle des activités de l'argininosuccinate synthétase, de l'argininosuccinase et de l'arginase á la naissance chez le rat
Authors: Husson A. , Vaillant R.
Published: December 2009

Some factors influencing the postnatal development of three urea cycle enzyme activities, argininosuccinate synthetase, argininosuccinase, and arginase, in the liver of newborn rats, are studied.Premature and postmature birth change the developmental pattern of these enzyme activities, suggesting that the increase in enzyme activity after birth is initiated by the birth process.Starvation of newborn animals after birth has no effect on the postnatal development of the three enzyme activities.Bilateral adrenalectomy at birth causes a partial inhibition of the postnatal increase of argininosuccinase and arginase activities while injection of cortisol prevents the effects of adrenalectomy.It is suggested that presence of adrenal secretion is one condition necessary for the increased activity of the prematures.
Subcellular distribution of lysophosphatidylinositol and lysophosphatidylcholine acyltransferases in rat brain
Authors: Pik J. R. , Thompson W.
Published: December 2009

Lysophosphatidylinositol acyltransferase utilizing arachidonoyl CoA and lysophosphatidylcholine acyltransferase utilizing linoleoyl CoA were measured in subcellular fractions of rat brain. In general, the distribution of activities paralleled that of NADPH–cytochrome c reductase. It is concluded that the endoplasmic reticulum is the major site of these acyltransferase activities in rat brain.
Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing
Authors: Daka N. J. , Laidler K. J.
Published: December 2009

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi–Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf−1/3, and the values extrapolated to infinite flow rate (vf−1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.
Metabolism of Δ24-sterols by yeast mutants blocked in removal of the C-14 methyl group
Authors: Pierce A. M. , Mueller R. B. , Unrau A. M. , Oehlschlager A. C.
Published: December 2009

We have investigated the metabolism of exogenously provided Δ24-sterols by whole cell cultures of a polyene-resistant mutant (D10) of Candida albicans blocked at removal of the C-14 methyl group. Comparison of the relative efficiencies of transmethylation at C-24 of selected sterol substrates revealed the following substrate preferences of the Candida Δ24-sterol methyltransferase (EC 2.1.1.41): zymosterol > 4α-methylzymosterol > 14α-methylzymosterol. Exogenous 4,4-dimethylzymosterol was not transmethylated by mutant D10. Incorporation of the 14C-labelled methyl group of S-adenosyl-L-[methyl-14C]methionine into the sterols of a D10 culture preloaded with zymosterol indicated that zymosterol was a better (40 ×) substrate than endogenous lanosterol. Feeding zymosterol to D10 and a polyene-resistant strain of Saccharomyces cerevisiae (Nys-P100) that was also blocked at removal of the C-14 methyl group gave 24-methyl sterols possessing Δ22 and ring B unsaturation. Mutant D10 was able to produce ergosterol from zymosterol whereas Nys-P100 produced ergosta-7,22-dienol. When grown in the presence of 3 μM 25-aza-24,25-dihydrozymosterol, a known inhibitor of the Δ24-sterol methyltransferase, Nys-P100 accumulated 14α-methylzymosterol, a minor metabolite in this mutant under normal growth conditions and hitherto unidentified as a yeast sterol.
Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography
Authors: Seligy Verner L.
Published: December 2009

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation–disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: [Formula: see text]. The order differs only slightly from the reverse of the desorption sequence, [Formula: see text]. Preferential interaction of H2A–H2B, H3–H4, and H2A–H2B–H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.
Spin-label study of the relation between enzymatic activity and lipid–protein organization in reconstituted cytochrome c oxidase
Authors: Denes A. S. , Stanacev N. Z.
Published: December 2009

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing < 20 μg lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipids such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.
Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa
Authors: Lin W. S. , Kapoor M.
Published: December 2009

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.
A rapid chromatographic method for the determination of ω-N-methylarginines in protein and muscle tissues
Authors: Zarkadas Constantinos G.
Published: December 2009

A rapid and sensitive chromatographic method is described for the complete separation and determination of NG-methylarginine, NG,NG-dimethylarginine, NG,N′G-dimethylarginine, and related compounds in protein and muscle tissue hydrolysates. This method is designed to be used both with conventional amino acid analyzers using a single column (10 × 0.9 cm) of Durrum type DC-6A resin at 51 °C, one buffer system (0.21 M sodium citrate pH 5.40), and one buffer flow rate (70 ml/h) and with the accelerated and fully automatic Beckman Spinco model 121M amino acid analyzer using a 10 × 0.28 cm microcolumn packed with the Beckman type AA-10 resin and operated with the same buffer at 7.9 ml/h. This procedure was successfully applied to the determination of ω-N-methylarginines in various animal tissues, and the presence of four as yet unidentified ninhydrin-positive compounds is reported.
Low density lipoprotein turnover in swine
Authors: Marcel Y. L. , Nestruck A. C. , Bergseth M. , Bidallier M. , Robinson W. T. , Jeffries D.
Published: December 2009

The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 ± 3.7, 19.7 ± 6.6, and 127 ± 70 h (mean of four studies).Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h−1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 ± 4.1 mg/h. Different kinetic models using two or three exponentials would provide different value s for the synthetic rate of apo-LDL.However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.
Parallelism between ethanol and imidazole interactions with horse liver alcohol dehydrogenase
Authors: Collins Patricia A. , Hanes Charles S. , Wong J. Tze-Fei
Published: December 2009

The rate effects of imidazole on the EE isoenzyme of horse liver alcohol dehydrogenase have been analysed in terms of the elucidated kinetic mechanism of the enzyme. These imidazole effects on both directions of the reaction within nonexcess as well as excess ranges of substrate concentrations pointed to the competition between imidazole and ethanol for binding to the same three enzyme species in the kinetic mechanism, namely the free enzyme, the enzyme–NAD+ complex, and the enzyme–NADH complex. Moreover, both imidazole and ethanol brought about an enhancement in the rate of dissociation of NAD+ from its binding site on the enzyme.
Acetylation of peptidyl-tRNA on rat liver polyribosomes
Authors: Green R. M. , Elce J. S. , Kisilevsky R.
Published: December 2009

Some properties of the known NH2-terminal acetylation of peptidyl-tRNA on rat liver polyribosomes were studied. Polyribosomes were incubated with [3H]acetyl-CoA and the products were separated into protein and peptidyl-tRNA fractions by an Ecteola-cellulose column procedure adapted for rapid, routine analysis of many samples, so that the characteristics of acetylation of the two fractions could be determined. Extraction of the polyribosomes with 0.5 M KCl–5 mM Mg(OAc)2 solubilized acetyltransferase activity which could catalyze histone acetylation, and the extract when added back to the extracted polyribosomes restored peptidyl-tRNA acetylation to 70% of the original values. The same extract could catalyze only a slight acetylation of peptidyl-tRNA free in solution isolated from polyribosomes by extraction with EDTA. These observations form the basis of an assay specific for the acetyl-CoA: peptidyl-tRNA N-acetyltransferase which is associated with rat liver polyribosomes.
Application of 125I radioimmunoassay to measure inhibition of precipitin reactions using carbohydrate-specific antibodies
Authors: Boullanger P. H. , Nagpurkar A. , Noujaim A. A. , Lemieux R. U.
Published: December 2009

Antibodies raised to an artificial antigen with β-D-galactopyranosyl groups as antigenic determinants were purified using an immunoadsorbent prepared from the hapten involved in the synthesis of the antigen. In order to study the specificity of these antibodies, 125I radiolabelling of either the artificial antigen or the antibody was used in the study of inhibitions of the precipitin reaction. The method, involving labelling of the artificial antigen and counting radioactivity in the supernatant, was found to be more accurate and faster than the usual methods based on measuring the amount of protein precipitated by chemical or spectroscopic methods.
Relationship between tissue levels of S-adenosylmethionine, S-adenosylhomocysteine, and transmethylation reactions
Authors: Hoffman Dennis R. , Cornatzer William E. , Duerre John A.
Published: December 2009

The concentrations of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and various methyltransferases were determined in the cerebrum, cerebellum, and liver of rats during development and aging. The liver contained from 3 to 7 and from 10 to 15 nmol AdoHcy per gram in young and adult rats, respectively. The AdoMet concentration was 60 to 90 nmol/g liver from rats of the same age and sex. It did not vary significantly with age. In the brain the AdoMet concentration was 45 to 50 nmol/g at birth and decreased to 20 nmol/g tissue with maturity of the organ. The level of AdoHcy in this organ was less than 1 nmol/g tissue throughout the life-span of the rat. Since the ratio of AdoMet to AdoHcy is relatively high, the rate of methylation of histones, DNA, or phosphatidylethanolamine in the liver or brain was not significantly influenced by AdoHcy. Under normal nutritional conditions, the tissue concentration of AdoMet is far above the Km values of histone and phosphatidylethanolamine methyltransferases. The levels of activity of these enzymes in liver and brain did not correlate with the cellular concentration of AdoHcy. The histone methyltransferase activity was elevated in rapidly proliferating tissues and declined markedly in the absence of histone biosynthesis. Phosphatidylethanolamine methyltransferase activity was elevated during development of the liver. The specific activity of the AdoHcy hydrolase remained relatively constant in the rat brain and liver. The activity of this enzyme was 10 times higher in liver than in brain, yet the concentration of AdoHcy was much lower in the latter organ. The tissue levels of this compound are evidently dependent on the rates of removal of homocysteine and adenosine. Adenosine deaminase was present in the liver and brain at relatively high concentrations, particularly during development.
Levels of glutathione in Escherichia coli
Authors: Loewen Peter C.
Published: December 2009

Log phase cells of Escherichia coli growing in minimal medium contain a basal level of glutathione (5 pmol/mL per Klett unit) which can increase more than sixfold when the cells reach stationary phase. Since the addition of cysteine alone to log phase cells illicits the same response, the increase in the intracellular pool of glutathione appears to be influenced by the amount of cysteine available for glutathione synthesis. Glucose depletion at low cell densities resulted in a decrease in the glutathione pool while the addition of amino acids other than cysteine did not affect the glutathione pool. Depletion of ammonia or proline as the nitrogen source also resulted in a decrease in the glutathione pool to one-third of the original basal level as did a shift to anaerobic growth. The large glutathione pool in stationary phase cells dropped from 31.5 to 4.5 pmol/mL per Klett unit within 30 min of transfer to fresh medium. There was no apparent correlation between changes in the glutathione and coenzyme A – glutathione disulfide (CoASSG) pools after a variety of metabolic disruptions.
On the characteristics of mitochondrial monoamine oxidase in pancreas and adipose tissues from genetically obese mice
Authors: Tong J. H. , D'Iorio A. , Kandaswami C.
Published: December 2009

The substrate specificity of mitochondrial monoamine oxidase (MAO) in pancreatic and adipose tissues of obese mice and their lean counterparts was determined. The pancreatic MAO of obese mice had a greater specific activity than that of the lean mice. The white adipose tissue MAO was found to be more active than the brown adipose MAO in both groups of mice. While there was no appreciable difference in the MAO activities of brown adipose tissues between obese and lean mice, the enzyme from the white adipose tissue of obese mice had a higher specific activity than that of the lean mice. The higher MAO activity in white adipose tissue was observed when tyramine or serotonin was employed as substrate but not with benzylamine. Examination of mitochondrial MAO from epididymal adipocytes revealed marked differences in the properties of the enzyme between whole adipose tissue and isolated adipocytes. The inhibition characteristics of MAO from these tissues were studied with the specific inhibitors clorgyline and deprenyl.
Protein kinase activity and ribosome phosphorylation in ethionine-treated rats
Authors: Treloar Margaret A. , Kisilevsky Robert
Published: December 2009

The regulation of protein synthesis at the level of the ribosome was investigated using the model system of ethionine-induced inhibition of protein synthesis. The phosphorylation of ribosomal protein S6 was examined in vivo during ethionine intoxication and during the adenine-induced reversal of ethionine intoxication. The extent of phosphorylation of S6 correlated well with protein synthetic activity observed after ethionine, and ethionine followed by adenine treatments. No clear correlation was observed in the ethionine system between cyclic adenosine 3′:5′-monophosphate concentration or the activity of ribosomal protein kinase and the phosphorylation of ribosomal protein S6. A role for a cyclic adenosine 3′:5′-monophosphate-dependent ribosomal phosphoprotein phosphatase is postulated.
Ammonia production and glutamine incorporation into glutathione in the functioning rat kidney
Authors: Welbourne T. C.
Published: December 2009

Incorporation of glutamine's γ-glutamyl moiety into glutathione was studied in the isolated perfused rat kidney under two conditions: (1) a resting state and (2) during reabsorption of a filtered glycylglycine load (designed to stimulate glutathione breakdown). Glutamine uptake and ammonia production were monitored simultaneously with [14C]glutamine (γ-glutamyl moiety) incorporation into glutathione.Renal glutathione content was highly dependent upon the presence of glutamine; in its absence, glutathione levels fell 40% in 30 min indicating synthesis does not keep pace with degradation. Presenting the kidney with a glycylglycine load doubled the rate of glutathione degradation. However, perfusing kidneys with physiological L-glutamine concentration (1 mM) maintained glutathione levels significantly above those in glutamine's absence in either condition 1 or 2. Evidence that glutamine functions as the γ-glutamyl donor in glutathione synthesis was established by the significant incorporation of [14C]glutamine into glutathione during condition 1 and the accelerated incorporation rate during glycylglycine loading (condition 2). Stimulating glutathione breakdown (condition 2) resulted in a significant increase in renal glutamine uptake and ammonia production apparently via the γ-glutamyltransferase pathway since inhibiting this activity blocked the incremental rise in glutamine uptake during glycylglycine loading. These results are consistent with the interpretation that stimulating γ-glutamyl cycle activity with a γ-glutamyl acceptor (glycylglycine) accelerates γ-glutamyl donor uptake (glutamine) and ammonia production via the γ-glutamyltransferase pathway.
Thermally induced changes in reconstituted and membranous cytochrome c oxidase
Authors: Denes A. S. , Stanacev N. Z.
Published: December 2009

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20–25 °C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16–18 °C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.
Circular dichroism studies of native and chemically modified Ca2+-dependent protein modulator
Authors: Walsh Michael , Stevens Frits C. , Oikawa Kimio , Kay Cyril M.
Published: December 2009

The structural features of the native Ca2+-dependent protein modulator and two chemically modified derivatives, namely, nitrotyrosyl modulator and alkylated modulator, were examined by circular dichroism. The binding of Ca2+ to the native molecule was accompanied by an increase in helical content from 40 to 49%, with little effect on the local environments of aromatic residues in the modulator. The Mg2+ and Mn2+ do not elicit the conformational change induced by the binding of Ca2+, which also stabilizes the modulator against urea denaturation.The overall secondary structure of nitrotyrosyl modulator is indistinguishable from that of the native protein and undergoes a similar conformational change upon binding Ca2+. These observations are in agreement with the fact that nitration has no effect on modulator functions. Futhermore, nitrotyrosyl modulator interacts with troponin I only in the presence of Ca2+, as detected by circular dichroism (cd). On the other hand, alkylation of five methionine residues on the modulator with benzyl bromide affects protein conformation, as evidenced by a reduced helical content of only 34%. Alkylated modulator retains the ability of the native protein to bind Ca2+ although the affinity of this derivative for Ca2+ is reduced some three orders of magnitude relative to the native protein, with Kd = 3.2 × 10−4 M.The results with the alkylated modulator, in conjunction with previous cd studies on N-chlorosuccinimide oxidized modulator are utilized to advance a model for the Ca2+ activation of modulator protein, based on three conformational states of the molecule.
Changes in the osmotic behaviour of phosphatidylcholine vesicles induced by interaction with polyamino acids
Authors: Chwang Willy K. , Pawagi Asha , Campbell Ian M.
Published: December 2009

The permeability of synthetic phosphatidylcholines has been studied with stopped-flow spectrometry by monitoring the initial shrinkage or swelling velocity of the sonicated vesicles. It was observed that aqueous diffusion was more rapid through dilinoleoyl- than through dioleoyl- which in turn was faster than through dipalmitoyl-phosphatidylcholine membranes. In contrast, when the vesicles were associated with poly-L-lysine, aqueous diffusion through dioleoyl- was greatly enhanced, whereas that through dilinoleoyl- and dipalmitoyl-phosphatidylcholine was diminished. When the vesicles were associated with polyglutamic acid, the permeability of dipalmitoyl decreased, that of dioleoyl increased, and there was little or no effect on dilinoleoyl vesicles. Measurements on the initial rate of optical density increase by dioleoyl- and dilinoleoyl-phosphatidylcholine vesicles, when they were mixed with polylysine solutions, showed that the rates of interaction by the two kinds of vesicles with the polypeptide were very different, dilinoleoyl reacting much more rapidly than dioleoyl. The observations indicate that phosphatidylcholine vesicles can interact with both acidic and basic polypeptides, and the reaction differs not only in relation to the charge on the polypeptide but also on the fatty acid composition of the lipid.
Spectral studies of human erythrocyte catalase
Authors: Palcic Monica , Dunford H. Brian
Published: December 2009

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as α helix, β pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.
Conformational comparison of (Val5, Trp8)-angiotensin II and (Val4, Trp7)-angiotensin III by fluorescence measurements
Authors: Schiller Peter W.
Published: December 2009

Intramolecular Tyr–Trp distances in the biologically active analogs (Val5, Trp8)-angiotensin II and (Val4, Trp7)-angiotensin III were determined by evaluation of singlet–singlet resonance energy transfer in dilute aqueous solution (3 × 10−5 M) at pH values of 1.5, 5.2, and 8.5. The higher transfer efficiencies obtained from donor (tyrosine) fluorescence quenching compared with those resulting from measurement of acceptor (tryptophan) fluorescence enhancement indicate the existence of additional quenching aside from energy transfer in both peptides. Taking into account additional quenching, similar Tyr–Trp distances in the range from 12.1 to 12.7 Å (1 Å = 0.1 nm) were obtained for the heptapeptide and the octapeptide at all three pH values. This finding provides evidence for a folding of the backbone in the C-terminal segment of both peptides. Furthermore, it can be concluded that titration of the carboxyl groups, of the histidine side chain, and of the α-amino group does not engender drastic changes in the overall conformation; however, subtle changes in intramolecular distance and fluorophore environment are observed. Small differences in conformational parameters are also apparent between (Val5, Trp8)-angiotensin II and (Val4, Trp7)-angiotensin III under identical conditions. The latter observation is of interest in view of the difference in specificity between angiotensin II and angiotensin III in their interaction with distinct classes of angiotensin receptors.
Diabetes: the pancreas and insulin, a retrospective view
Authors: Levine Rachmiel
Published: December 2009

not available
The effects of prostaglandins on secretion of glucagon and inselin by the perfused rat pancreas
Authors: Akpan J. O. , Hurley M. C. , Pek S. , Lands W. E. M.
Published: December 2009

The secretion of both glucagon and insulin by the isolated perfused rat pancreas was significantly stimulated by 10−7 M PGH2. Experiments to show that the stimulated secretion was mediated by conversion of PGH2 to TXA2 or TXB2 revealed no correlation between the amount of secretion and the amount of thromboxane formed. Conversion of PGH2 with a crude platelet thromboxane synthase preparation caused a progressive loss of ability to secrete insulin, whereas the capacity to stimulate release of glucagon remained at about one-half the maximal level. This relatively stable and selective secretogogue action on the α-cells appeared to be due to the formation of PGD2 by the platelet preparation. Direct administration of PGD2 confirmed this interpretation and showed clearly that this prostaglandin is a potent secretagogue for glucagon with little activity in stimulating the release of insulin. Our results have shown high and relatively equal stimulation of secretion by α- and β-cells with exogenous PGE2, PGF2α, and PGH2, little or no secretion by either cell type with TXA2, TXB2, or PGI2, and a unique selective stimulatory action of PGD2 upon the α-cell.
Microtubules, colchicine, and lymphocyte blastogenesis
Authors: Rudd Christopher E. , Rogers Kem A. , Brown David L. , Kaplan J. Gordin
Published: December 2009

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10−6 M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor–ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by α-methyl mannoside (αMM) and by colchicine do not exclude the possibility that the latter compound may act partly by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of αMM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the αMM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.
Chick pineal serotonin acetyltransferase: a diurnal cycle maintained in vitro and its regulation by light
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

We have reproduced in vitro the diurnal cycles in levels of serotonin acetyltransferase activity found in the chick pineal gland in vivo. The more closely the lighting conditions of culture matched those under which the birds were raised, the closer was the similarity between cycles in levels of enzyme activity in vitro and in vivo. Repetitive cycles in levels of acetyltransferase activity persisted in culture for at least 4 days under a diurnal cycle of illumination, and at least 2 days in continuous darkness. When glands were explanted into culture in the light phase of a cycle, short periods of further exposure to light markedly stimulated subsequent increase of acetyltransferase in the dark (after a short lag). Prolonged exposure to light in culture markedly inhibited increase of enzyme activity. Cycles in the levels of enzyme activity in glands cultured under altered light cycles were regulated primarily by changes in illumination. However, the endogenous biological 'clock' remained at least partly entrained to the original light cycle. Increase of acetyltransferase activity in vitro was markedly stimulated by theophylline plus compound Ro. 20.1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) under all lighting conditions. Kinetics (to the time of attaining maximum levels in situ) of the increase under diurnal lighting and in constant darkness were indistinguishable from those in vivo. A high concentration of dl-propranolol markedly stimulated an increase in acetyltransferase activity in glands cultured in constant darkness but had little effect on glands under diurnal lighting or continuous illumination.
Purification and properties of a citrate-binding transport component the C protein of Salmonella typhimurium
Authors: Sweet G. D. , Somers J. M. , Kay W. W.
Published: December 2009

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1–2.6 μM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.
Electron microscopy of serum amyloid protein in the presence of calcium; alternative forms of assembly of pentagonal molecules in two-dimensional lattices
Authors: Pinteric L. , Painter R. H.
Published: December 2009

The P component is a protein believed to be of serum origin commonly found in the highly ordered proteinaceous tissue deposits called amyloid. Both the protein recovered from the tissue and its serum counterpart have an identical characteristic appearance in the electron microscope, consisting of pentagonal plates which are often assembled as columns of stacked discs. These images, seen in the absence of calcium, are replaced by three more complex assemblies when low concentrations of calcium are present. One of the structures is crystalline (III), the other two appear to be planar lattices, one having a distinguishable structure with threefold symmetry (I). The structural elements of the other lattice (II) which takes the form of a cylinder are less distinct. It is suggested that the regular arrays which P component forms in the presence of calcium may be the basic framework on which amyloid deposits form.
The amino acid sequence of porcine intestinal calcium-binding protein
Authors: Hofmann Theo , Kawakami Michiko , Hitchman Anthony J. W. , Harrison Joan E. , Dorrington Keith J.
Published: December 2009

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometry analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.
Lysozyme catalysis: kinetics of the hydrolysis of cell wall oligosaccharides
Authors: Boekelheide Kim , Patt Steven L. , Weisz Greg , Baldo John H. , Sykes Brian D.
Published: December 2009

The cleavage of cell wall tetrasaccharide, the β(1 → 4)-linked dimer of the basic repeating disaccharide N-acetyl-D-glucosamine-β(1 → 4)-N-acetyl-D-muramic acid, by lysozyme has been studied at various concentrations of lysozyme and over long time ranges. A theoretical analysis of the kinetic results indicates that direct hydrolysis of the tetrasaccharide by binding in sub-sites CDEF of the active site of lysozyme is significant at long times relative to the transglycosylation pathway. The binding constant for tetrasaccharide in CDEF is shown to be 103 times larger than that determined on the basis of an analysis of kinetic data over a more restricted range of times and concentrations.
The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III
Authors: Takahashi Seishi , Hours Christian , Chu Alan , Denhardt David T.
Published: December 2009

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes ([Formula: see text], fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, λp rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with λp rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of [Formula: see text] replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from [Formula: see text]-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.
Effect of heparin on the metabolism of putrescine in vivo
Authors: Sourkes Theodore L. , Missala Krystyna
Published: December 2009

Rats and guinea pigs which are given heparin metabolize intraperitoneally injected [14C]-putrescine to 14CO2 at reduced rates. The results have been considered in relation to the heparin-induced liberation of diamine oxidase from tissues into the blood stream.
The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes
Authors: Mitranic M. M. , Sturgess J. M. , Moscarello M. A.
Published: December 2009

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialyltransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60–80 μg WGA to the Golgi membrane, while only 40 μg of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialyltransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20–30 min in the presence of 1 μmol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 × 10−6 M from a Steck–Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 × 10−5 M/mg of Golgi membrane protein.