bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of rat liver
Authors: Bag Jnanankur , Sells Bruce H.
Published: December 2009

Nonpolysomal cytoplasmic mRNA protein complexes (free mRNP) from normal and regenerating rat liver were isolated by affinity chromatography on oligo (dT)-cellulose column. The mRNP complexes bound to the oligo(dT) cellulose were eluted in a three-step process by using low ionic strength buffer (25 mM Tris–HCl, pH 7.5) at (i) 2 °C, (ii) 37 °C, and (iii) containing 50% formamide. The protein content of these three mRNP fractions, calculated from their buoyant densities, was found to be 50, 63, and 78%, respectively.Sixteen polypeptides of Mr 31 000 to 90 000 were present in these mRNP fractions. These polypeptides were found to be of nonribosomal origin. The polypeptide complements of these mRNP fractions were qualitatively similar when both major and minor components were considered. Considerable differences in the major polypeptides of the different mRNP fractions were observed. The major polypeptides of the mRNP eluted at 2 °C (mRNP-1) were of Mr equal to 51 000, 65 000, 74 000, 78 000, 80 000, 84 000, 85 000, and 90 000. Five additional polypeptides of Mr equal to 44 000, 46 000, 49 000, 60 000, and 64 000 were present as major components in mRNP eluted at 37 °C (mRNP-2). On the other hand, the relatively protein-rich mRNP fraction eluted with 50% formamide (mRNP-3) consists of only four major polypeptides of Mr equal to 49 000, 51 000, 57 000, and 66 000. The polypeptides present in mRNP-1 and mRNP-2 of both normal and regenerating rat liver were similar. However, the mRNP-3 fraction of the regenerating rat liver contains an additional polypeptide of Mr equal to 54 000.
Horseradish peroxidase. XXXIV. Oxidation of compound II to I by periodate and inorganic anion radicals
Authors: Nadezhdin A. , Dunford H. B.
Published: December 2009

The one-electron oxidation of horseradish peroxidase compound II to compound I by sodium periodate was observed. The bimolecular rate constant for the NaIO4–compound II interaction is equal to 9.5 ± 1 × 10−3 M−1 s−1 at room temperature. Irradiation, using ultraviolet light, of the solution containing compound II and persulfate in the presence of bicarbonate, chloride, or bromide, leads to the fast accumulation of compound I due to the oxidative action of [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] anion radicals, which are products of the photolysis.
Studies on isocitrate lyase isolated from Lupinus cotyledons
Authors: Vanni P. , Vincenzini M. T. , Nerozzi F. M. , Sinha S. P.
Published: December 2009

Isocitrate lyase (threo-Ds-isocitrate glyoxylate-lyase, EC 4.1.3.1) was isolated from cotyledons of Lupinus seedlings, purified 100-fold with respect to its initial specific activity and characterized (Km, pH optimum, Mg2+ requirement, sulfhydryl inhibitors, and synthase activity). The final purified preparation consisted of two homogeneous protein bands clearly separated by electrophoresis on polyacrylamide gel and chromatography on Sephadex G 200.Reducing agents are necessary for the maintenance of enzyme activity. The most effective reducing agent studied was 1,4-dithioerythreitol. The effect of several metabolites (oxalate, malonate, phosphoenolpyruvate, succinate, malate, tartrate, gluconate-6-phosphate, sorbose, sorbitol, and inositol) on the activity of purified preparations was tested. Oxalate proved to be the strongest inhibitor, seconded closely by phosphoenolpyruvate. The spectral characteristics of the purified enzyme are as follows: ultraviolet peak at 280 nm and fluorescence peak at 340 nm. The solid state infrared spectrum of the enzyme (lyophilized) showed that the enzyme was mostly in the α-helix conformation with very slight random orientation.
The effects of alcohols on the structure of human glycophorin
Authors: Byers David M. , Verpoorte Jacob A.
Published: December 2009

The effects of alcohols on human glycophorin were monitored by circular dichroism, solvent perturbation of absorption spectra, fluorescence of 8-anilino-1-naphthalene sulfonate, and sedimentation equilibrium in the ultracentrifuge. Both ethanol and 2-chloroethanol gradually increase the α helix in glycophorin and its sialic acid free counterpart. The same alcohols do not cause a cooperative transition in the structure of the polypeptide chain of glycophorin. Other alcohols also increase the α-helix content of glycophorin. Binding of ANS to glycophorin is abolished at relatively low alcohol concentrations. Ethanol at 60% (v/v) reduces the molecular weight ratio of glycophorin and at the same time increases the exposure of tyrosine residues to solvent. These observations indicate a complex mechanism of interaction of weakly protic solvents with this stable membrane protein.
Biosynthèse des glycoconjugués pulmonaires. III. Mannosylation des accepteurs lipidiques et protéiniques
Authors: Levrat Christiane , Louisot Pierre
Published: December 2009

Earlier studies on the biosynthesis of mannosyl glycoproteins in microsomes of rat lungs have been extended to show that a mannosyltransferase was able to catalyze the incorporation of mannose from GDP-mannose. In sheep lungs, we have shown that a mannosyltransferase activity is involved at microsomal level This enzymatic system is able to catalyze the incorporation of mannose from GDP-mannose into three different endogenous acceptors. The enzymatic products are as follows: a mannolipid (product A) extracted with CHCl3–CH3OH (2:1), mannose is also incorporated into a product B extracted with CHCl3-CH3OH–H2O (10:10:3), and into another product C insoluble in water and precipitable in trichloracetic acid. The kinetic studies of product biosynthesis suggest that there does not exist a precursor–product relationship. The product A is synthesized in larger quantities than the products B and C. Exogenous dolichoi-monophosphate stimulates the incorporation of [14C]mannose into mannolipid (product A) only. The results indicate that the mannolipid (product A) is not an intermediate in the transfer of mannose from GDP-mannose to the other products (B and C). The product A of the reaction, the mannolipid, has the properties of a polyprenyl-phosphoryl-mannose. This is illustrated by its lability to dilute acid but stability to dilute alkali at low temperature, and by its mobility on thin layer chromatography. However, this product A is not similar to dolichyl-monophosphoryl-mannose from pig liver.
N-Acylation of tyramines: purification and characterization of an arylamine N-acetyltransferase from rat brain and liver
Authors: Yu Peter H. , Boulton Alan A.
Published: December 2009

The N-acylation of tyramine isomers and other biogenic amines has been studied. The liver exhibits the highest activity towards tyramines, while the brain exhibits a low but significant activity. In the brain, tyramine N-acylation activity was heterogenously distributed. The arylamine N-acetyltransferase has been partially purified from both rat liver and brain, the two enzymes being quite similar with respect to their chromatographic properties, optimal pH requirement (pH 7.8), and their kinetic parameters. The product N-acetyltyramine is not oxidized by liver amidohydrolase or monoamine oxidase.
The kinetic parameters of trehalase in whole and disrupted mitochondrial preparations from two insects with asynchronous muscle
Authors: Brandt N. R. , Hurlburt K. L. , Huber R. E.
Published: December 2009

The kinetic parameters of trehaiase in honey bee and flesh fly mitochondria were compared. The studies were carried out with whole mitochondria and with mitochondria disrupted in various ways and to different degrees. Honey bee mitochondrial trehalase was significantly activated by Lubrol WX treatment (30.0-fold), by high pH treatment (20.8-fold), and by a treatment consisting of 10 passes through a French press (37.9-fold) but not by the other treatments tried (salt, proteases, Waring blender, and sonication), despite the fact that these treatments also disrupted the mitochondria significantly. The activation effect was on the Vmax. The Km value did not change. Simple breakage of either the outer or inner (or both) membranes was not sufficient to activate trehalase from honey bees, which showed that the activation was not an indirect result of a change in the ease with which trehalose can pass through the membranes. Honey bee trehalase is the first trehalase from insects with asynchronous muscle which has been shown to be activatable by physical and chemical methods. Flesh fly mitochondrial trehalase behaved quite differently from the honey bee enzyme in that it could not be activated by any of the techniques tried, even when there were significant amounts of disruption.
Studies on the neutral glycosphingolipids of porcine plasma
Authors: Clarke J. T. R. , Stoltz J. M.
Published: December 2009

Swine are widely used as models for studies on atherosclerosis because the composition and metabolism of their plasma lipoproteins are similar to those in man. As part of studies on the role of glycosphingolipids (GSL) in plasma lipoprotein metabolism, the neutral GSL of porcine plasma were extracted and analyzed. The structures of the GSL oligosaccharides were determined by permethylation analysis. In addition to the four major species previously reported (glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide, and N-acetylgalactosaminylgalactosylgalactosylglucosylceramide), significant amounts of galactosyl-ceramide and digalactosylceramide were also found. The findings are interpreted as indicating that the composition of porcine plasma GSL may be subject to considerable intrinsic, probably genetic, variability.Analyses of various classes of plasma lipoproteins isolated by sequential preparative ultracentrifugation showed that 55–80% of the GSL occurs in the d 1.020–1.060 and d 1.090–1.210 g/mL fractions, as it does in man. However, unlike in human plasma, in which the bulk of the GSL occurs in the d 1.020–1.060 g/mL fraction, the majority of the GSL in porcine plasma is about equally distributed between the two fractions. In both species, the distribution tends to parallel the distribution of total cholesterol and phospholipid among the major lipoprotein fractions.
Erratum: Chemical synthesis of sn-3-phosphatidyl sulfocholine, a sulfonium analog of lecithin
Authors: Tremblay Paul-Alain , Kates Morris
Published: December 2009

not available
Erratum: Lysyl dipeptide and tripeptide model systems for racemization studies in amino acid and peptide chemistry
Authors: Benoiton N. Leo , Kuroda Keiji , Cheung S. T. , Chen Francis M. F.
Published: December 2009

not available
Changes induced by cold adaptation in the brown adipose tissue from several species of rodents, with special reference to the mitochondrial components
Authors: Ricquier D. , Mory G. , Hemon P.
Published: December 2009

(1) The effects of cold adaptation upon the brown adipose tissue have been studied in rats, hamsters, mice, and guinea pigs.(2) Striking effects were found for total tissue as well as at the mitochondrial level, e.g., increases in protein and phospholipid contents, changes in phospholipid fatty acid composition (a decrease in the percentage of palmitic and palmitoleic acids and an increase in stearic and linoleic acids), and a change in the mitochondrial polypeptide composition (a marked increase in a 32 000 molecular weight polypeptide, except for hamsters).(3) In situations where animals exhibit a greatly enhanced capacity for nonshivering thermo-genesis (cold adaptation for rats, mice, and guinea pigs, birth for guinea pigs, and hibernation ability for hamsters, dormice, and garden dormice), brown fat mitochondria are characterized by the occurrence of large amounts of the 32 000 molecular weight polypeptide characteristic of these mitochondria.
Components and mechanism of action of ATP-driven proton pomps
Authors: Alfonzo Marcelo , Racker Efraim
Published: December 2009

We have studied the composition of ATP-driven proton pumps from bovine heart mitochondria and have reconstituted the oligomycin-sensitive ATPase complex from its individual components. The complex contains 9 to 10 subunits of which 5 are assembled in the soluble F1 protein, 2 are required for the attachment of F1 to the membrane and 2 form the proton channel within the membrane. With the help of information obtained from studies of the chloroplast and the bacterial proton pumps, we can tentatively assign a function to each of the subunits of the pump. The position of F1 outside of the membrane seen in electron micrographs of negatively stained preparations, does not appear to be an artifact. Evidence from immunological studies, chemical derivatizations as well as further electron microscopy (positive staining and freeze–etching), support this statement. We describe in this paper a 28 000-dalton polypeptide which has been isolated from the mitochondria membrane and is required for the reconstitution of oligomycin-sensitive ATPase and 32Pi–ATP exchange activity. We propose a mechanism of action of the proton pump in which the key energy-yielding reaction is the binding of Mg2+ to the protein. The function of the proton gradient is to displace Mg2+ from this site to permit cyclic repetition of the binding process. Essential for this scheme is the cyclic opening and closing of the proton channel. We have outlined our present approaches to test this hypothesis.
The action of tributyltin chloride on the uptake of proline and glutamine by intact cells of Escherichia coli
Authors: Singh A. P. , Bragg P. D.
Published: December 2009

Tributyltin chloride inhibits growth and uptake of glutamine and proline into intact cells of Escherichia coli. It causes efflux of the accumulated amino acids. A pH gradient generated in intact cells and everted membrane vesicles is dissipated by this compound. These effects do not require lipoic acid but are dependent on the presence of chloride, bromide, or iodide ions. We conclude that tributyltin chloride can catalyse a transmembrane OH−–anion exchange reaction and that this is its mode of inhibition of the uptake of these amino acids. The response of proline and glutamine uptake to the inhibitor is similar and is consistent with the transport of both amino acids requiring an electrochemical gradient of protons.
Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria
Authors: Arron Geoffrey P. , Edwards Gerald E.
Published: December 2009

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(β-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.
Erratum: Renal adaptation to phosphate deprivation in the Hyp mouse with X-linked hypophosphatemia
Authors: Tenenhouse H. S. , Scriver C. R.
Published: December 2009

not available
Inhibition of electron transport and oxidative phosphorylation in plant mitochondria by gladiolic acid and structurally-related aromatic ortho dialdehydes
Authors: White G. A. , Elliott W. B.
Published: December 2009

Gladiolic acid (GA, 4-methoxy-5-methyl-o-phthalaldehyde-3-carboxylic acid), an antifungal aromatic ortho dialdehyde produced by Penicillium gladioli was found to be a potent inhibitor of electron transport and oxidative phosphorylation reactions in sweet potato and mung bean mitochondria. Similar results were also found with the naturally occurring ortho dialdehydes, cyclopaldic acid, quadrilineatin, and flavipin as well as the synthetic dialdehydes, 3-formyl opianic acid and o-phthalaldehyde. Because of their highly reactive ortho-diformyl grouping, GA and structurally related dialdehydes apparently act as multisite inhibitors affecting electron transport and oxidative phosphorylation (at each coupling site). Gladiolic acid has no uncoupling effect like 2,4-dinitrophenol and does not have the same point of interaction in the energy transfer process as oligomycin. Several "partial" reactions of phosphorylation (Mg+2–DNP-stimulated ATPase; ATP–Pi exchange) were strongly inhibited by the various dialdehydes. Flavipin and quadrilineatin are potent inhibitors (80% at a concentration of 25 μM) of site III phosphorylation. Gladiolic acid and related ortho dialdehydes inactivate the catalytic activity of native cytochrome c in vitro. Lysyl ε-NH2 rich cytochrome c may be a major site of GA action in the intact mitochondrion. In view of the high chemical reactivity of the ortho-diformyl group, it is suggested that mitochondrial function may be affected by aromatic ortho dialdehydes through a combination of reactions involving cross-linking of amino groups on membrane polypeptides and monofunctional reaction with free amino groups important for enzyme function, including ε-NH2 groups on cytochrome c. Cross-linking in mitochondrial membrane systems might affect function by interfering with molecular motion in the operation of the terminal portion of the electron-transport chain. The primary toxicological mode of action of GA and related dialdehydes appears to be due to inhibition of mitochondrial function.
Protein phosphorylation and the proliferation of chick embryo fibroblasts: analysis by in vitro phosphorylation using isolated plasma membranes and whole cell homogenates
Authors: Branton Philip E.
Published: December 2009

The relationship between plasma membrane and whole cell protein phosphorylation and chick embryo fibroblast proliferation was studied using an in vitro labeling technique employing [γ-32P]ATP and isolated plasma membranes or whole cell homogenates. Cultures containing proliferating cells in log phase or containing cells stimulated to proliferate by the addition of serum were compared with cultures in which proliferation was inhibited due to cell density. Cell proliferation was found to be associated with increased plasma membrane basal protein kinase activity, but decreased membrane and whole cell cyclic AMP-dependent kinase activity. The phosphorylation of a number of membrane and whole cell polypeptides differed between proliferating and density-inhibited cells, suggesting that these phosphoproteins may be involved in the regulatory events associated with cell proliferation. Of interest, however, was the fact that the phosphoprotein differences found with serum-stimulated and sparse, rapidly dividing cells were generally not the same. These observations suggest that the protein phosphorylation events associated with rapid proliferation of sparse, log-phase cells may differ from those involved in serum-activated proliferation of quiescent cells.
The isolation and properties of a ribonuclease associated with influenza virus
Authors: Arora D. J. S. , Vincent L. , Hill-Schubert J.
Published: December 2009

A ribonuclease activity associated with influenza virus has been purified to homogeneity. This preparation is free of DNAase, phosphodiesterase, and phosphatase activities. The purified enzyme has a pH optima of 7.0 at 37 °C, and moves as a single band on sodium dodecyl sulfate – polyacrylamide gel with an estimated molecular weight of 84 000.
Occurrence of globulin-like migrating blood albumins, or GLIMBAL, in pathological rat and human sera
Authors: Silvestrini B. , Natali P. G. , Catanese B. , Barillari G. , Cordiali-Fei P.
Published: December 2009

The presence in the serum of albumins having a globulin-like electrophoretic mobility (globulin-like migrating blood albumins or GLIMBAL) has been determined by Immunoelectrophoresis in rat and human sera. GLIMBAL have been detected occasionally (14%) in rat sera during the acute inflammation induced by yeast injection. GLIMBAL were, on the other hand, present in pathological human sera from patients with rheumatoid arthritis (50%), systemic lupus erythematosus (69%), and liver disease (17%). These data indicate that the occurrence of GLIMBAL in human sera is more frequent than previously described.Many pathological conditions are accompanied by electrophoretic changes of serum, consisting of an increase of globulins and eventually albumin decrease; our data indicate that these changes may in some conditions reflect a process of albumin transformation rather than an increase in globulin synthesis.
Effect of phosphoenolpyruvate carboxykinase inhibition on renal metabolism of glutamine: in vivo studies in the dog and rat
Authors: Vinay P. , Coutlée F. , Martel P. , Lemieux G. , Gougoux A.
Published: December 2009

The effect of in vivo administration of mercaptopicolinate (MCP), a potent inhibitor of phosphoenolpyruvate carboxykinase (PEPCK) and of gluconeogenesis, on renal ammonia production was studied in dog and rat. In the dog, MCP depressed only slightly renal ammonia production, but increased strikingly renal glutamine extraction. Aspartate and alanine synthesis by the kidney were also considerably enhanced. The renal tissue metabolite profile showed an accumulation of oxaloacetate and malate but glutamate concentration was decreased. In the rat, MCP depressed renal glutamine extraction but did not abolish ammonia production in a proportionate fashion. Thus other amino acids support ammoniagenesis during PEPCK inhibition. The renal metabolite profile indicated inhibition of gluconeogenesis at the PEPCK step. It is concluded that in both species renal ammonia production can proceed through "non-PEPCK-dependent" pathways in vivo, at least during PEPCK inhibition.
Impairment by covalent modification of the ability of Phaseolus vulgaris phytohemagglutinin to stimulate cultured human lymphocytes: a comparison of different forms of covalent modification
Authors: Dupuis Gilles , Clairoux-Moreau Sylvie , Chevrier Pierre
Published: December 2009

Phytohemagglutinin (PHA) isolated from Phaseolus vulgaris has been modified by treatment with various chemical reagents and the modified proteins have been tested for their ability to stimulate peripheral lymphocytes from two healthy human donors, in vitro. Reaction of PHA with citraconic anhydride, S-methyl isothiourea, or 2-hydroxy-5-nitrobenzyl bromide produced derivatives which retained the ability to stimulate lymphocytes, at low concentrations. Acylation of the lectin with acetic anhydride or masking of the carboxyl side chains by reaction with glycinamide–carbodiimide impaired stimulation. When PHA was treated with N-bromosuccinimide or with tetranitromethane, the derivatives were ineffective as lymphocyte stimulants. Chemical modifications affected, in some cases, the quaternary structure of the lectin. Glycinamide-, homoarginine-, and nitro-PHA were tetramers whereas acetyl-, citraconyl-, and N-bromosuccinimide-treated lectin were dimers. Antinative lectin antiserum cross-reacted with all the modified proteins, except in the case of the N-bromosuccinimide derivative. The results show that, in the human lymphocyte transformation assay, the mitogenic property of PHA may depend on intact aspartic, glutamic, and tyrosine residues whereas lysine residues do not appear to be essential.
Structure and composition of the alkali-insoluble cell wall fraction of Coprinus macrorhizus var. microsporus
Authors: Bottom Carey B. , Siehr Donald J.
Published: December 2009

The alkali-insoluble (R-) fraction from the cell walls of Coprinus macrorhizus var. microsporus is a highly branched glucan, containing α-(1 → 4), β-(1 → 3), and β-(1 → 6) linkages as shown by methylation, partial acid hydrolysis, and enzymic hydrolysis. The α-(1 → 4)-linked segments are joined by occasional β-(1 → 3) links as suggested by the identification of 2-O-α-glucopyranosyl erythritol in the hydrolysate of the reduced, periodate-oxidized glucan. Hydrolysis of the permethylated glucan gave nearly equimolar amounts of 2,4-di- and 2,3-di-O-methyl-D-glucose. Methylation analysis of the residue from enzymic hydrolysis, the "CORE-fraction," indicated the presence of glucose residues in this fraction linked through positions O1, O3, O4, and O6. Hydrolysates of the R-fraction contained mannose, glucosamine, and amino acids in addition to glucose.
Membrane electricity as a convertible energy currency for the cell
Authors: Skulachev V. P.
Published: December 2009

The role of transmembrane electric potential difference (Δψ) in mitochondria, chloroplasts, and bacteria has been considered. Since the electric capacitance of membranes is much lower than the pH buffer capacitance of water phases, Δψ proves to be the primary form of energy produced by generators of electrochemical H+ potential difference [Formula: see text]. There are 11 distinct types of [Formula: see text]-generating systems in coupling membranes, involved in respiratory and light-dependent electron and proton transfer, as well as in ATP and PPi hydrolysis and synthesis. Bacteriorhodopsin is the simplest [Formula: see text] generator. However, even in this case, the molecular mechanism of Δψ production remains obscure. Many types of work can be supported by [Formula: see text] with no ATP involved so that [Formula: see text] proves to be not only a transient intermediate of oxidative and photosynthetic phosphorylation but also a convertible energy currency for the cell. Among the [Formula: see text]-supported activities, mechanical work was recently demonstrated. It can be exemplified by the motility systems of (i) flagellar bacteria and (ii) blue–green algae. As was found in multicellular cyanobacteria, [Formula: see text] can be used for a power transmission over distances as long as 1 mm. It seems to be probable that in large cells of eukaryotes (e.g., in muscle fibers) giant mitochondria may serve as power-transmitting structures. Na+–K+ gradients can be used to stabilize [Formula: see text] in bacteria. It is suggested that the primary function of unequal distribution of these cations between the microbial cell and the medium is [Formula: see text] buffering.
pH-Dependent amino acid induced conformational changes of rabbit muscle pyruvate kinase
Authors: Kwan Chiu-Yin , Davis Robert C.
Published: December 2009

The interactions of L-Phe and L-Ala with rabbit muscle pyruvate kinase depended upon the nature of divalent metal ions studied: Mg(II), Co(II), Mn(II), and Ni(II). L-Phe inhibited all metal derivatives of the enzyme except Mn(II)–enzyme. L-Ala inhibited only Ni(II)–enzyme and had no effect on other metal derivatives. The inhibition by L-Phe could be partially or completely reversed by L-Ala for all metal derivatives. The mode of inhibition of pyruvate kinase by L-Phe depended upon pH as well as the nature of activating divalent metal ions. The sigmoidal response increased with increasing pH for all metal derivatives inhibited by L-Phe. L-Phe and L-Ala strongly perturbed the coordination sphere of enzyme bound Co(II), but not Ni(II). There were poor correlations between visible circular dichroic (cd) spectral changes and the corresponding kinetic changes. However, L-Phe and (or) L-Ala induced ultraviolet cd and difference absorption spectral changes, on the other hand, corresponded remarkably well with the kinetic observations.
Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase
Authors: Kwan Chiu-Yin , Gabriel Jerome L. , Davis Robert C.
Published: December 2009

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.
Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors
Authors: Roncari Daniel A. K. , Wang Henry , Desai Kappu S.
Published: December 2009

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.
Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by use of aminoacyl-tRNA synthetases as phylogenetic probes
Authors: Kwok Yau , Wong J. Tze-Fei
Published: December 2009

The cross-species reactivities between tRNAs and aminoacyl-tRNA synthetases have been employed as a basis to estimate the relatedness of various prokaryotes to the eukaryotes. The tRNA of Halobacterium cutirubrum, unlike that of other prokaryotes tested, including Agrobacterium tumefaciens, Arthrobacter luteus, Bacillus subtilis, Bacillus stearothermophilus, Escherichia coli, Micrococcus luteus, Myxococcus xanthus, Rhodopseudomonas spheroides, and Thermus aquaticus, was found to share with yeast, rat liver, and wheat germ tRNA a distinct preference for aminoacylation by eukaryotic synthetases from yeast as opposed to prokaryotic synthetases from either E. coli or R. spheroides. These results suggest that phylogenetically H. cutirubrum is more closely related to the eukaryotes than to the eubacteria.
Inhibition of purified wheat germ DNA dependent RNA polymerase by pyran copolymer
Authors: Greene Robert , Munson Benjamin
Published: December 2009

The inhibition of DNA and RNA polymerases in vitro by pyran copolymer has been shown to be related to its affinity for divalent cations. The present investigation was designed to explore further the nature of this inhibition using completely purified eukaryotic RNA polymerase II from wheat germ. Inhibition was determined as a function of divalent ion concentration and, as previously seen with less pure enzyme preparation, was greatest at low (Mn2+) and least at higher concentrations. No inhibition was observed at concentrations greater than 4.8 mM MgCl2 in the presence of 10 μg pyran/mL. The inhibition by pyran copolymer was exerted immediately unlike other polyanions, such as heparin and polynucleotides. This indicates that it stops RNA chain growth immediately as do known chelators of divalent cations. The size of pyran copolymer was shown to affect the extent of inhibition when different sized polymers were fractionated from a heterogenous single lot. However, when sized fractions were obtained from different lots we could not show a size-dependent inhibition. Although the mechanism by which pyran copolymer exerts its biological effect is unknown, it may well be related to its association with cations. The inhibition of enzymes requiring these cations appears to be a sensitive method of observing such an association.
The kinetic properties of phosphoenolpyruvate carboxykinase of Escherichia coli
Authors: Krebs Anita , Bridger William A.
Published: December 2009

Initial rate kinetic studies on phosphoenolpyruvate carboxykinase of Escherichia coli are consistent with either of two sequential kinetic mechanisms: rapid equilibrium random, or partially ordered. However, the kinetics of isotope exchange at chemical equilibrium allows clear discrimination in favor of a random mechanism containing a preferred pathway of substrate addition and product release. All plots of exchange rates vs. reactant concentrations leveled off at constant rates at saturating levels of substrates; since complete inhibition of any exchange was not observed, a compulsory sequence may be eliminated. High concentrations of phosphoenolpyruvate and (or) ATP repressed the oxaloacetate [Formula: see text] bicarbonate exchange, however, indicating that the latter pair of substrates add most favorably to the free enzyme. Exchange rates between various substrate–product pairs were not identical, ruling out a formally rapid equilibrium random mechanism with rate-limiting interconversion of the central complex. A comparison of the relative rates of the overall reaction and the ATP-dependent oxaloacetate [Formula: see text] bicarbonate exchange showed the latter to be much faster than net formation of oxaloacetate. The requirement for ATP in promoting this exchange may be rationalized in terms of substrate synergism, with occupation of the ATP binding site a requisite for its catalysis.
Glycosaminoglycans of tissues of the domestic fowl
Authors: Kwai Hang Kwet Fane Ng , Anastassiadis Phoebus A.
Published: December 2009

Mucopolysaccharide–peptide complexes (MPS-P) from skin, comb, wattle, liver, kidney, spleen, lung, heart, ovaries, egg yolk, infundibulum, magnum, isthmus, shell gland, and vagina of the White Leghorn laying hens were isolated after extraction with acetone, papain hydrolysis, and cetyl pyridinium chloride precipitation. They were analyzed for characteristic components of the glycosaminoglycan (GAG) and carbohydrate parts of glycoproteins and for amino acids. The infrared spectra of the MPS-P were studied for detection of the GAG present. The MPS-P were submitted to cellulose acetate electrophoresis for detection of GAG and to column chromatography on an anionic resin for quantitative determination of GAG.
Polypeptide turnover in brown adipose tissue mitochondria during acclimation of rats to cold
Authors: Himms-Hagen Jean , Dittmar Elizabeth , Zaror-Behrens Gloria
Published: December 2009

During the initial phase of cold-induced growth of brown adipose tissue in rats there is a selective increase in the incorporation of infused [3H]phenylalanine into mitochondrial membrane polypeptides of molecular weight 25 000–35 000. This is interpreted as a selective increase in the synthesis of a 32 000 polypeptide, of which the proportion is known to increase in brown adipose tissue mitochondria when the tissue has a high capacity for thermogenesis, as in the cold-acclimated rat. This polypeptide is known to be associated with the thermogenic proton conductance pathway. A simultaneous selective decrease in degradation or the formation from larger mitochondrial membrane polypeptides may also occur. In fully cold-acclimated rats, in which a new steady state is reached, there is a general increase in turnover of all mitochondrial membrane polypeptides but no marked selective changes in pattern of incorporation of radioactive amino acid or in rates of disappearance of radioactivity from groups of polypeptides. Isolated brown adipose tissue mitochondria incorporate [3H]phenylalanine principally into polypeptides of molecular weight 25 000–35 000. No change in the pattern of incorporation occurred in mitochondria isolated from brown adipose tissue of cold-exposed (2 weeks) rats. On the basis of these and preceding results it is concluded that the cold-induced change in mitochondrial composition in brown adipose tissue, which occurs at the same time as tissue and mitochondrial growth, is brought about by selective changes in cytosolic protein synthesis and possibly also by selectively altered degradation or conversion of mitochondrial polypeptides.
The effect of polypeptide–lipid interactions on the ultraviolet spectrum of the olefinic bonds of the lipid
Authors: Campbell Ian M. , Pawagi Asha B.
Published: December 2009

The effect of associating acidic and basic polypeptides with dilute suspensions of vesicles composed of various unsaturated phospholipids was assessed with regard to optical density and ultraviolet absorption. Associating basic polypeptides with phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol vesicles, or acidic polypeptide with phosphatidylcholine vesicles, caused an increase in the optical density of the preparations, with no measurable effect on the intensity of the ultraviolet spectrum of the olefinic bonds of the lipid. Associating basic polypeptides with phosphatidylcholine vesicles, in addition to causing similar increases in optical density, resulted in a large decrease in the intensity of ultraviolet absorption by the olefinic bonds. This implies that the interaction between the basic polypeptides and phosphatidylcholine vesicles results in major alterations in the microenvironment of the olefinic bonds, which would require intimate association of the polypeptide with the ninth carbon region of the acyl chains. These observations support the conclusion, drawn from our earlier studies, that the association of basic polypeptides and liquid crystalline phase phosphatidylcholine vesicles is peculiar in that it involves a major hydrophobic component.
Guanosine metabolism in Neurospora crassa
Authors: Greer W. L. , Pendyala L. , Wellman A. M.
Published: December 2009

Two aspects of guanosine metabolism in Neurospora have been investigated, (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.
Antifreeze proteins from the shorthorn sculpin, Myoxocephalus scorpius: isolation and characterization
Authors: Hew Choy L. , Fletcher Garth L. , Ananthanarayanan V. S.
Published: December 2009

The antifreeze proteins (AFP) of the shorthorn sculpin, Myoxocephalus scorpius, were isolated and compared with the AFP of the winter flounder. The shorthorn sculpin was found to contain one major and one minor antifreeze protein with an approximate molecular weight of 10 000 – 11 000 in the winter. The major AFP was isolated and characterized. It had many characteristics in common with the flounder AFP studied in our laboratories. These characteristics included its seasonal appearance, size, amino acid composition, the abundance of alanine in the composition, the extent of antifreeze activity, and the nature of the secondary structure. It is suggested that both the sculpin and flounder AFP are structurally homologous and belong to the same type of polypeptide antifreeze.
Mannose containing glycopeptides of cells derived from human teratocarcinoma (line PA 1)
Authors: Rasilo Maija-Liisa , Wartiovaara Jorma , Renkonen Ossi
Published: December 2009

Human teratocarcinoma derived cells, line PA 1, maintained in the undifferentiated state, yielded upon exhaustive pronase digestion unusually large glycopeptides (fraction A), which showed on gel filtration an apparent molecular weight larger than 7400. These glycopeptides derived from whole cell proteins carried large-sized oligosaccharides as evidenced by repeated pronase treatments, hydrazinolysis, and β-elimination experiments. The oligosaccharides consisted of mannose, fucose, galactose, and N-acetylglucosamine.The PA 1 cells contained also oligomannosyl type glycans, presumably linked to asparagine (fraction C glycopeptides). These glycopeptides were strongly bound to Con A – Sepharose and their oligosaccharides were released by endo-β-N-acetylglucosaminidase H. The liberated glycans ranged from Man5GlcNAc to Man9GlcNAc as analyzed by paper chromatography."Pulse–chase" experiments suggest that there is a precursor–product relationship between the mannose label in the fraction C (oligomannosyl type) glycopeptides and the fraction A glycopeptides.
Relative suitability of 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glycerylphosphorylcholine and different acyl donors for phosphatidylcholine synthesis via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes
Authors: Holub B. J. , Plekarski J. , Possmayer F.
Published: December 2009

The relative suitability of the 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glyceryl-phosphorylcholine and different acyl donors were tested as substrates for phosphatidylcholine synthesis via the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes. The acyl acceptor was an almost equi-molar mixture of the [3H]palmitoyl plus [14C]stearoyl species of 1-acyl-sn-glycero-3-phosphorylcholine with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, or arachidonoyl-CoA serving as the acyl donor. At all concentrations of acyl acceptor, reaction velocities with 20:4-CoA ≥ 18:2-CoA > 18:1-CoA > 16:0-CoA > 18:0-CoA. Furthermore, the acyltransferase selectively utilized the 1-palmitoyl over the 1-stearoyl species of 1-acylglycerylphosphorylcholine by 4.2- to 5.7-fold under optimal assay conditions with the various acyl-CoA thiolesters. However, the degree of preference exhibited for the 1-palmitoyl-sn-glycero-3-phosphorylcholine, as acyl acceptor, versus the 1-stearoyl homologue with palmitoyl-CoA as the acyl donor was not significantly different from that obtained with the other acyl-CoA derivatives. Thus, the specificity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase indicates an ability of this enzyme to produce dipalmitoyl phosphatidylcholine but it cannot independently explain the predominance of dipalmitoyl phosphatidylcholine in lung or the tendency of stearate at the 1-position to associate with fatty acids of increasing unsaturation at the 2-position.
Inhibition of renal gluconeogenesis and phosphoenolpyruvate carboxykinase activity by 3-mercaptopicolinic acid: studies in rat, guinea pig, dog, rabbit, and man
Authors: Watford Malcolm , Vinay Patrick , Lemieux Guy , Gougoux André
Published: December 2009

The inhibition of gluconeogenesis in isolated kidney tubules by 3-mercaptopicolinate, a specific inhibitor of the phosphoenolpyruvate carboxykinase reaction, is species specific. At any given concentration of the inhibitor, the degree of inhibition is in the order rat > guinea pig ≥ man > rabbit ≥ dog. The intracellular distribution of PEPCK activity (as measured in both the forward and backward directions) supports the conclusion that in the intact cell the cytosolic enzyme is preferentially inhibited. Analysis of the inhibition failed to demonstrate any significant differences between the enzymes, regardless of intracellular or animal source. All enzymes exhibited a similar Ki (2–10 μM) for 3-mercaptopicolinate when assayed in the forward direction. The observed preferential cytosolic inhibition could be due to either in situ differences in metallic ions concentration or a restriction in mitochondrial permeability for mercaptopicolinate.
Thyroid hormone regulation of translation in tadpole tail muscle
Authors: Saleem Mohammed , Atkinson Burr G.
Published: December 2009

Recent in vivo and in vitro studies with polyribosomes from the tail muscle of T3-treated tadpoles establish that this hormone initiates a regulating effect on tadpole tail muscle which operates at the translational level and results in an overall decreased rate of protein synthesis (Saleem, M. &Atkinson, B. G. (1978) J. Biol. Chem. 253, 1378–1384). This hormone-induced decrease in the rate of protein synthesis is partially, if not wholly, due to the presence of a sarcoplasmic factor(s) inhibiting ribosomal translational efficiency. This research employs the use of a reconstituted, cell-free polypeptide synthesizing system as a means to substantiate the presence of an inhibitor and further elucidate the mechanism by which this inhibitory factor(s) depresses protein synthesis. The results of this study further demonstrate the presence of an inhibitor of protein synthesis in the tail muscle sarcoplasm of T3-treated tadpoles and suggest that this depressed synthetic activity results from an interaction of the inhibitor with ribosomal or polyribosomal constituents.
Jack bean urease (EC 3.5.1.5). I. A simple dry ashing procedure for the microdetermination of trace metals in proteins. The nickel content of urease
Authors: Dixon Nicholas E. , Blakeley Robert L. , Zerner Burt
Published: December 2009

A simple and inexpensive procedure for determination of microgram quantities of metal ions in proteins is described and tested with nickel and iron. The method involves (a) dry ashing in an oxygen atmosphere at 450–460 °C in Pyrex vessels, (b) conversion of the metal oxides or other compounds to readily soluble species, and (c) spectrophotometric analysis. An improved procedure for the direct spectrophotometric determination of nickel using dimethylglyoxime is accurate to ± 2% or better with samples of 1–5 μg of nickel. These techniques were used to determine that the nickel content of freshly prepared jack bean urease is 2.00 ± 0.12 g-at./96 600 g protein. This corresponds to 2.0 nickel ions per subunit. This result was confirmed by atomic absorption analysis, which also showed that calcium, manganese, cobalt, and iron are not present in significant amounts in urease.