bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of magnesium and polyamines on the reactivity of individual ribosomal subunit proteins to lactoperoxidase-catalyzed iodination
Authors: Michalski Chester J. , Boyle Stephen M. , Sells Bruce H.
Published: December 2009

30S and 50S subunits, in the presence of either 20 mM Mg2+ or 6 mM Mg2+ and 5 mM spermidine plus 25 mM putrescine, were observed to completely associate to form 70S monosomes as monitored by sucrose gradient sedimentation. Subunits maintained under the above ionic conditions were compared with 30S and 50S particles at low (6 mM) magnesium concentration with respect to the reactivity of individual ribosomal proteins to lactoperoxidase-catalyzed iodination. Altered reactivity to enzymatic iodination of ribosomal proteins S4, S9, S10, S14, S17, S19, and S20 in the small subunit of ribosomal proteins, L2, L9, L11, L27, and L30 in the large subunit following incubation with high magnesium or magnesium and polyamines suggests that a conformation change in both subunits accompanies the formation of 70S monosomes. The results further demonstrate that the effect of Mg2+ on subunit conformation is mimicked when polyamines are substituted for magnesium necessary for subunit association.
Synthesis and turnover of sulfogalactoglycerolipid, a membrane lipid, during spermatogenesis
Authors: Kornblatt M. J.
Published: December 2009

The synthesis and turnover of sulfogalactoglycerolipid (SGG) were studied by in vivo labelling of SGG with 35S. The loss of [35S]SGG from the testes and its appearance in the vas deferens plus epididymis were followed with time. DNA was labelled by administration of [3H]thymidine and the behavior of the two isotopes was compared. The results demonstrate that SGG synthesis occurs only in very early spermatocytes and that, once made, the compound does not turn over. The SGG is lost from the testes when germinal cells die or mature into spermatozoa.
The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds
Authors: Percy Maire E. , Chang Lebe , Demoliou Catherine , Baumal Reuben
Published: December 2009

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.
A rapid method for the purification of antibody–enzyme conjugates
Authors: Pagé M. , Audette M. , Caron M.
Published: December 2009

A method is described for a rapid and efficient separation of enzyme-labelled antibodies from the free enzyme following the coupling reaction. A single passage of the reaction mixture on a protein A – Sepharose CL-4B column gave a sharp separation of the free enzyme from the conjugate.
Isolation and translation in vitro of poly(A)+ RNA from the free-living nematode Panagrellus silusiae
Authors: Noble J. Scott , Pasternak J.
Published: December 2009

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)− RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)− RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate – polyacrylamide gels. Poly(A)− RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.
Characteristics of glutamine metabolism by rat kidney tubules: a carbon and nitrogen balance
Authors: Vinay Patrick , Lemieux Guy , Gougoux André
Published: December 2009

The metabolism of glutamine by a suspension of rat kidney tubules was studied in vitro. The influence of duration of incubation, glutamine concentration, and metabolic state of the donor animals was investigated. The relative importance of glucose synthesis, amino acid production, and oxidation to CO2 was estimated by drawing a complete balance of the nitrogens and the carbon chains of the extracted glutamine. It was found that the initial (first 15 min) rate of glutamine utilization was significantly greater than the subsequent rate due to an initial, but transient, extracellular accumulation of glutamate. This phenomenon was suppressed when a small amount of glutamate was added to the incubation medium. Glucose production constitutes the major fate for glutamine metabolism. No net oxidation of glutamine could be detected with 1 mM glutamine during the first 30 min. However, glutamine oxidation becomes significant after prolonged incubation (16% at 120 min). The metabolic fate of glutamine differs when 5 or 10 mM are presented to the tubules, glutamate production and oxidation to CO2 becoming more important. Metabolic acidosis or a 48-h fast increases glutamine extraction and enhances its utilization towards glucose synthesis while they depress glutamate accumulation and oxidation to CO2. Metabolic alkalosis has the opposite effect. It is concluded that the metabolism of glutamine in vitro is dependent on the conditions of the study. Furthermore, total oxidation to CO2 is not a major fate for glutamine metabolism at physiological concentration and is not enhanced by acidosis in the rat kidney in vitro.
Inhibition of carboxypeptidase A catalyzed peptide hydrolysis by 3-phenylpropanoate at activating and nonactivating substrate concentrations
Authors: Poorman Roger , Kuo Maria , Johnson Douglas I. , Lin Sharon , Sebastian John F.
Published: December 2009

The carboxypeptidase A catalyzed hydrolyses of five structurally related dipeptide substrates in the presence of the inhibitor 3-phenylpropanoate have been studied. At nonactivating substrate concentrations, 3-phenylpropanoate is a mixed inhibitor of carbobenzoxyglycyl-L-phenylalanine hydrolysis and a noncompetitive inhibitor of the hydrolyses of benzoylglycyl-L-phenylalanine, cinnamoylglycyl-L-phenylalanine, hydrocinnamoylglycyl-L-phenylalanine, and acetylglycyl-L-phenylalanine. When carbobenzoxyglycyl-L-phenylalanine and benzoylglycyl-L-phenylalanine exhibit substrate activation, inhibition by 3-phenylpropanoate is mixed but appears to be mostly competitive. Proposed here is a site for the binding of 3-phenylpropanoate along with a kinetic mechanism consistent with these data.
Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide
Authors: Bundle David R.
Published: December 2009

An artificial antigen was prepared from 4-O-β-D-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.
The Interactions of adenylates with allosteric citrate synthase
Authors: Talgoy Michael M. , Duckworth Harry W.
Published: December 2009

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, α-ketoglutarate, can compete with OAA in the absence of KClbut not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.
Phospholipids of the differentiating bacterium Caulobacter crescentus
Authors: Jones Darrell E. , Smith Joseph Donald
Published: December 2009

The phospholipid composition of the stalked and swarmer cell types of the differentiating, Gram-negative bacterium Caulobacter crescentus was determined. The phospholipid composition of the stalked cell type was 86.5% phosphatidylglycerol, 10.4% lysylphosphatidylglycerol, and 3.0% cardiolipin; that of the swarmer cell type was 84.1, 11.4, and 4.4%, respectively. Phosphatidylethanolamine, which is a major phospholipid component of most Gram-negative bacteria, was totally absent.
Effects of ribosomal wash factors and spermidine on endogenous and exogenous mRNA stimulated protein synthesis in the wheat germ cell-free system
Authors: Elahi Elahe , Jones George H.
Published: December 2009

Differential effects of Mg2+, spermidine, and reticulocyte ribosomal wash factors on the translation of endogenous, myeloma, and globin mRNA's have been observed in studies with the wheat germ cell-free protein synthesizing system. Spermidine stimulated globin mRNA translation but not the translation of endogenous wheat germ messages, and the polyamine actually inhibited the translation of myeloma mRNA. Ribosomal wash factors, on the other hand, stimulated endogenous and myeloma mRNA dependent protein synthesis in an Mg2+-dependent fashion but inhibited globin mRNA translation. The combination of ribosomal wash factors and spermidine was either stimulatory or inhibitory depending on the Mg2+ concentration and the message. It was further observed that translation of exogenous myeloma mRNA proceeded for only 60 min at 25 °C under all conditions tested in this study, while translation of endogenous wheat germ messages continued for longer periods of time. No differential effects of spermidine on the synthesis of high molecular weight myeloma proteins were observed.
Dr. Best, insulin, and molecular evolution
Authors: Jukes Thomas H.
Published: December 2009

In this introductory paper of the special issue of the Canadian Journal of Biochemistry dedicated to the memory of the late Charles H. Best, Thomas Jukes describes the atmosphere of Dr. Best's laboratory, reminisces about Dr. Best and his research, and then discusses the central role of insulin as a model protein in the study of molecular evolution.
Insulin receptors in transformed fibroblasts and in adipocytes: a comparative study
Authors: Maturo III J. M. , Hollenberg M. D.
Published: December 2009

This study compares the receptor for insulin in rat adipocytes with an insulin receptor from transformed AL/N mouse embryo fibroblasts (SVS AL/N). In many respects the receptors from the two tissue sources are very similar. Crude solubilized receptor preparations from both fat cells and SVS AL/N cells exhibit complicated insulin-binding kinetics (two binding plateaus) as well as an apparent chromatographic heterogeneity (Sepharose 6B); each receptor preparation yields two insulin-binding components having apparent Stokes radii of about 7.2 and 3.8 nm. In contrast, the receptors isolated from both cell types by affinity chromatography behave as single chromatographic components (Sepharose 6B) having apparent stokes radii of 3.8 nm and exhibit simple insulin-binding kinetics without evidence for receptor–ligand co-operativity. The isolated receptor from SVS AL/N cells (KD about 1.2 nM) possesses a slightly lower insulin affinity than does the receptor from fat cells (KD about 0.8 nM); nonetheless, the SVS AL/N receptor exhibits an appropriate ligand specificity for insulin and insulin analogues. Receptors from both cell types are similarly sensitive to trypsin and exhibit an identical dependence on pH for insulin binding. Differences in the two receptor preparations are observed in the effect of NaCl and phospholipase C, reagents which augment insulin binding in adipocyte membranes but do not affect the binding by SVS AL/N cell membranes. Additionally, iodoacetamide, N-ethylmaleimide, and ethanol markedly reduce the binding by SVS AL/N membranes, whereas the binding by adipocyte membranes is unaffected. It is concluded that certain differences between the receptors from the two sources may be attributed to differences in the membrane environment in which the receptors reside. However, in view of the many similarities between the two receptor preparations, it is suggested that the same receptor macromolecule may participate in the response of a variety of cell types not only to insulin but also to other insulin-like polypeptides.
A model for the study of the oral administration of peptide hormones
Authors: Saffran Murray , Franco-Saenz Roberto , Kong Ann , Papahadjopoulos Demetrios , Szoka Francis
Published: December 2009

The intragastric administration of lysine vasopressin (LVP) to rats is used as a model to study the biological activity of orally administered peptide hormones. Using a modification of the antidiuretic assay of Sawyer, LVP given by stomach tube caused a significant antidiuresis that was dose dependent in doses of 300 to 2000 mU. The simultaneous administration of the protease inhibitor, Trasylol®, increased the antidiuretic effect of LVP. The synthetic peptide (1-deamino, 4 valine)-8-D-arginine-vasopressin also caused a dose-dependent prolonged and significant antidiuresis. No pressor effect was observed after intragastric administration of LVP in doses up to 40 U/rat. We are now using this model to test other procedures for enhancing the activity of lysine vasopressin administered in the gastrointestinal tract such as encapsulation into liposomes. The information gained with vasopressin will then be applied to insulin with the ultimate goal of making oral administration practical.
The influence of dietary choline intake upon liver ethanolamine metabolism
Authors: Tokmakjian S. , Haines D. S. M.
Published: December 2009

The effect of dietary choline upon liver ethanolamine metabolism was investigated by measurement of the changes in the liver levels of phospholipids and their precursors and in the metabolism of intraportally administered [14C]ethanolamine which occur when choline feeding is restored to 3-day choline deficient rats. The earliest detectable actions of choline were restoration of the decreased liver phosphatidyl choline level and beginning decreases in the elevated levels of liver ethanolamine, ethanolamine phosphate, CDP-ethanolamine, and phosphatidyl ethanolamine. The impairment at the CTP:ethanolaminephosphate cytidylyltransferase (EC 2.7.7.14) reaction induced by choline deficiency was reversed more slowly than were the increased levels of the ethanolamine-containing intermediates.In a further experiment it was observed that the liver levels of all of the ethanolamine-containing compounds were progressively diminished when the content of dietary choline was increased up to 1.2%. These findings indicate that exogenous choline exerts an effect upon liver metabolism which causes decreases in the liver levels of ethanolamine and its derivatives, including phosphatidyl ethanolamine. The nature of this action of choline is not known. In an experiment where rats were fed a diet containing 1% ethanolamine, the pattern of increase in the levels of the liver ethanolamine-containing substances did not mirror the pattern found in choline deficiency. This suggests that the increases in liver ethanolamine compounds caused by dietary choline deficiency are not due primarily to an excessive accumulation of free ethanolamine.
Low density lipoprotein binding, internalization, and degradation in human adipose cells
Authors: Angel A. , D'Costa M. A. , Yuen R.
Published: December 2009

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell – lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025–1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 ± 0.9 μg LDL protein/mL and a Vmax of 15–80 μg LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F− inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d < 1.006) and high density lipoprotein (HDL) (d = 1.075–1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.
Chemical synthesis of sn-3-phosphatidyl sulfocholine, a sulfonium analog of lecithin
Authors: Tremblay Paul-Alain , Kates Morris
Published: December 2009

A sulfonium analog of lecithin has been reported to replace the ubiquitous phosphatidyl choline in a non-photosynthetic diatom, Nitzschia alba. The structure of this sulfonium analog has now been established by chemical synthesis using the following methods: (i) condensation of sn-3-phosphatidic acid (dimyristoyl-, dipalmitoyl-, distearoyl-, and dioleoyl-) with sulfocholine chloride in the presence of triisopropylbenzenesulfonylchloride in chloroform–pyridine (9:1); and (ii) phosphorylation of 1,2-dipalmitoyl-sn-glycerol with monophenylphosphoryl-dichloridate followed by a reaction with sulfocholine in the presence of pyridine and finally removal of the blocking phenyl group by catalytic hydrogenolysis. The desired synthetic products were obtained in overall yields of 50–70% and 11% for methods (i) and (ii), respectively, and were characterized by elemental analyses; infrared spectroscopy, nuclear magnetic resonance spectrometry, and mass spectrometry; optical rotation; and thin-layer chromatography mobilities. Comparison of the synthetic analogs with the natural sulfolecithin showed them to be identical, except for the nature of the fatty acid chains, thus establishing the natural product as sn-3-phosphatidyl sulfocholine.
Translocation of isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Translocation of membrane-bound isomeric (5-, 12-, and 16-doxyl) spin-labelled radioactive cytidinediphosphodiglycerides (CDP-diglycerides) from guinea pig liver microsomal membranes to mitochondrial membranes was studied. When microsomal membranes containing known amounts of isomeric spin-labelled radioactive CDP-diglycerides were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by microsomal contamination, indicating that translocation of labelled CDP-diglycerides from microsomal to mitochondrial membranes had occurred. The rate of loss of paramagnetism in microsomal and in reisolated mitochondrial membranes was found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. When reisolated mitochondria containing translocated isomeric spin-labelled radioactive CDP-diglycerides were further incubated with sn-3-glycerophosphate, the formation of labelled phosphatidylglycerol was detected. Data from these studies established that the translocation of labelled CDP-diglycerides from microsomal membranes to both outer and inner mitochondrial membranes had occurred.This study established that the isolated guinea pig liver mitochondria are capable of biosynthesis of polyglycerophosphatides (phosphatidylglycerolphosphate, phosphatidylglycerol, and diphosphatidylglycerol or cardiolipin) but depend on the microsomal supply of CDP-diglyceride, an obligatory precursor in the formation of polyglycerophosphatides. This liponucleotide can be translocated, as shown here, to outer and inner mitochondrial membranes for further biosynthetic utilization.
Dicarboxylic acid transport in Escherichia coli K12: involvement of a binding protein in the translocation of dicarboxylic acids across the outer membrane of the cell envelope
Authors: Bewick Mary A. , Lo Theodore C. Y.
Published: December 2009

We have previously found that the dicarboxylate transport system in Escherichia coli K12 is an active transport system and that at least one binding protein and two cytoplasmic membrane transport components are involved in the uptake of dicarboxylic acids. Recently, through surface labelling studies, some dicarboxylate binding proteins were found to be exposed on the cell surface. In the present paper, we demonstrate that the dicarboxylate transport component located in the outer membrane can be inactivated by two different kinds of nonpenetrating inhibitors, viz. proteases, and diazosulfanilic acid. These inhibitors seem to act on the dicarboxylate binding protein. By adding this protein to inactivated cells or to transport-negative mutants, we have succeeded in reconstituting the dicarboxylate transport system. These findings suggest that the dicarboxylate binding protein found on the cell surface plays an essential role in the translocation of dicarboxylic acids across the outer membrane.
Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1
Authors: Chan P. K. , Liew C. C.
Published: December 2009

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate – polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.
Stimulation of glucose transport in rat adipocytes by calcium
Authors: Taylor Wayne M. , Hau Lena , Halperin Mitchell L.
Published: December 2009

Glucose transport in rat adipocytes was studied by monitoring the conversion of [1-14C]-glucose to 14CO2 in a system where glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent which rapidly reoxidizes NADPH. Calcium increased both basal and insulin-stimulated apparent rates of glucose transport by approximately 40%. The maximum velocity of the apparent rate of glucose transport was increased by extracellular calcium both in the presence or absence of insulin. There was no change in the glucose concentration required for half-maximal rates of 14CO2 production. Calcium also enhanced the stimulation of apparent rates of glucose transport by insulin when examined over a range of hormone concentrations. Adipocyte cAMP concentrations were significantly lowered by calcium under conditions which led to increased apparent rates of glucose transport. In contrast, cobalt and nickel, antagonists of calcium action, elevated adipocyte cAMP levels and inhibited apparent rates of glucose transport. Agents which inhibit transmembrane calcium flux (verapamil, tetracaine, and procaine) inhibited apparent rates of glucose transport despite a reduction in adipocyte cAMP concentration.On the basis of the above data we suggest that calcium may increase apparent rates of glucose transport in rat adipocytes both by lowering intracellular cAMP concentration and by a further mechanism independent of changes in the level of cAMP. These results are consistent with the hypothesis that glucose transport in rat adipocytes may be controlled, in part, by a cAMP-induced phosphorylation mechanism.
Structural homologies in alanine-rich acidic ribosomal proteins from procaryotes and eucaryotes
Authors: Visentin Louis Peter , Yaguchi Makoto , Matheson Alastair T.
Published: December 2009

The amino acid composition and amino-terminal sequence have been determined for the alanine-rich, acidic ribosomal 'A' protein (equivalent to Escherichia coli L7/L12) from three procaryotic cell types that live under extreme environmental conditions (Arthrobacter glacialis, Clostridium pasteurianum, and Bacillus stearothermophilus) as well as from wheat germ, a eucaryote source. These data are compared with previously published 'A' protein sequences from other procaryotes and eucaryotes. All the procaryotic 'A' proteins, with the exception of the very acidic 'A' protein from Halobacterium cutirubrum, show similar charge, size, and amino acid composition, as well as an extensive sequence homology in the N-terminal region. Some differences are observed between gram-negative and gram-positive bacteria. The 'A' proteins from eucaryotes contain two tyrosine molecules, an amino acid absent in procaryotic 'A' proteins, as well as a reduced number of valine residues and an increased amount of aspartic acid. The N-terminal sequence of wheat germ 'A' protein shows considerable homology with other eucaryotic 'A' proteins and also with H. cutirubrum. It also shows some sequence homology with E. coli 'A' proteins.
The interaction between beef cardiac troponin T and troponin I as demonstrated by ultraviolet absorption difference spectroscopy, circular dichroism, and gel filtration
Authors: Hincke M. T. , McCubbin W. D. , Kay C. M.
Published: December 2009

The specific interaction of bovine cardiac troponin T with troponin I has been demonstrated at a 1:1 molar ratio by absorption difference spectroscopy, near and far ultraviolet circular dichroism, and gel filtration chromatography. The maintenance of the sulfhydryl groups of both proteins in the reduced state was essential in order to demonstrate interaction between cardiac troponin I and troponin T using the aforementioned methodology. Carboxamidomethylated troponin I and troponin T samples were prepared by reaction with iodoacetamide. Spectrophotometric titration of the two proteins with 2-chloromercurinitrophenol and amino acid analysis of their carboxamidomethylated derivatives revealed that cardiac troponin I possesses two cysteine residues while cardiac troponin T has one. The modified troponin T possesses properties identical to those of the native molecule. The modification of troponin I is accompanied by an increase in secondary structure and a loss in ability to interact with troponin T at 0.5 M NaCl ionic strength. However, at 0.3 M NaCl the modified troponin I was shown by gel filtration chromatography to interact very weakly with troponin T. On the other hand, the modified troponin I interacts with troponin C in a manner identical to the native protein, indicating that the troponin T interaction domain of the molecule is distinct from that region which interacts with troponin C.
Analysis of the surface topography of glycogen phosphorylase a: implications for metabolic interconversion and regulatory mechanisms
Authors: Fletterick R. J. , Sprang S. , Madsen N. B.
Published: December 2009

Computer drawings of the van der Waals contours of atoms on the surface represent the phosphorylase molecule at 2.5 Å (1 Å = 0.1 nm) resolution with color coding for acidic and basic residues, bound ligands, or conformational changes. The asymmetry resulting from the twofold axis of each dimer provides two faces which can be distinguished structurally and functionally. Thus, a concave catalytic face contains the glycogen storage sites on its periphery with entrances to the glucose-1-phosphate binding sites of the active centers, adjacent to the pyridoxal phosphate moieties, near its center. Just outside the active site is a binding site for negative effectors such as caffeine. On the side of the dimer opposite to the catalytic face is found a convex control face containing the binding sites for the allosteric activator AMP, for which ATP also competes. Quite close to these sites are found the Ser-14-phosphates hydrogen bonded to Arg-69 and Arg-43′ of the symmetry-related monomer. Each Ser-14-phosphate is surrounded by positive charges, including more than are found on adjacent sequences, and therefore, comparative studies on peptides cannot describe fully the specificity and binding requirements of the kinase and phosphatase.The surface topography of the glucose stabilized T state (taut) and the changes which occur during the allosteric transitions induced by AMP and substrates are discussed in terms of their functional implications for the control of both the intrinsic enzymic activity and of the metabolic interconversions between the a and b forms. In particular, the two conformational states (taut and relaxed) exhibit different structural arrangements of the binding sites for the negative effector, caffeine, and the positive effector, AMP. Linkage between the various effector sites is demonstrated by conformational changes in the surface topography. Since the control face is opposite to the catalytic face, the interconverting enzymes can bind to, and act on, the phosphorylase while the latter is bound to glycogen. The Ser-14 residues are only 40 Å apart across the control face and could be bridged readily by the multimeric kinase (α4β4γ4δ4, 1 300 000 daltons) or the large molecular weight form of the phosphatase. The T →R conformational change causes the Ser-14-phosphate to move 5 Å in from the surface, which may be related to the 20-fold decrease of the phosphatase Vm with unchanged Km.
Methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase from porcine liver Location of the activities in two domains of the multifunctional polypeptide
Authors: Tan L. U. L. , MacKenzie R. E.
Published: December 2009

Chymotryptic cleavage of the trifunctional protein methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase from pig liver yields a fragment of two-thirds the original polypeptide that retains only synthetase activity. A smaller polypeptide corresponding to about one-third of the original polypeptide was shown earlier to retain dehydrogenase–cyclohydrolase activity. On immunodiffusion, the synthetase fragment cross-reacts and shows partial identity with antibodies raised against the uncleaved enzyme but shows nonidentity with the dehydrogenase–cyclohydrolase fragment, suggesting that the two fragments are derived from different regions of the polypeptide. Amino-terminal analysis of the peptides and uncleaved enzyme indicate that the dehydrogenase–cyclohydrolase activities are located at the amino-terminal region and the synthetase near the carboxyl-terminal portion of the polypeptide.
Distinctive properties of glutamine synthetase from the cyanobacterium Anacystis nidulans
Authors: Emond Diane , Rondeau Normand , Cedergren R. J.
Published: December 2009

The intracellular levels of glutamine synthetase (GS) in Anacystis nidulans grown under different conditions were determined using a whole-cell assay. Nitrate-grown cells have 64% more GS than cells grown in ammonium sulfate. Nitrogen starvation does not affect GS levels appreciably. Incubation of nitrate-grown cells with ammonium sulfate does not change the ratio of γ-glutamyl transferase activities stimulated by Mg2+ and Mn2+ ions. An in vitro test of adenylylation indicates that algae do not have an endogenous adenylyl transferase (ATase) and that algal GS is not adenylylatable by the Klebsiella aerogenes ATase. Some characteristics of the GS–membrane complex were determined by centrifugation of the complex under varying conditions of pH and ionic strength. In this way, it was shown that acid pH (4.5) stabilizes the complex and high ionic strength tends to solubilize the enzyme. A simple partial purification of GS (89-fold) was developed based on the sedimentation properties of GS.
Cyanobacterial evolution: results of 16S ribosomal ribonucleic acid sequence analyses
Authors: Bonen L. , Doolittle W. F. , Fox G. E.
Published: December 2009

We report here the sequences of oligonucleotides released by T1-ribonuclease digestion of the 16S ribosomal RNA's (rRNA's) of the unicellular cyanobacteria Agmenellum quadruplicatum (strain BG-1) and Synechococcus 7502. We compare them with sequences previously obtained for the 16S rRNA's of six other cyanobacteria and two chloroplasts, and conclude that: (i) Synechocystis-like unicells form a discrete cluster which also (and surprisingly) includes Agmenellum quadruplicatum, usually considered to be a Synechococcus; (ii) filamentous cyanobacteria of the genera Nostoc and Fischerella arose from within the Synechocystis group; (iii) phylogenetic diversity (and hence presumably evolutionary antiquity) within the Synechococcus group is very great; and (iv) red algal chloroplasts are of definite cyanobacterial origin, while Euglena chloroplasts are of separate and quite possibly noncyanobacterial origin. We also present the results of a computer-aided search among the 10 oligonucleotide 'catalogues' for families of related but nonidentical sequences. Examination of these families reinforces the above conclusions.
Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components
Authors: Iatrou Kostas , Gedamu Lashitew , Dixon Gordon H.
Published: December 2009

Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly two of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.
Roles of noradrenaline and protein synthesis in the cold-induced increase in purine nucleotide binding by rat brown adipose tissue mitochondria
Authors: Desautels Michel , Himms-Hagen Jean
Published: December 2009

Exposure of a rat to cold (4 °C) is known to induce a biphasic change in brown adipose tissue mitochondria, believed to reflect alterations in the thermogenic, purine nucleotide sensitive proton conductance pathway; an initial rapid and large increase in purine nucleotide binding, unaccompanied by any marked change in the 32 000 polypeptide which is the binding site for these nucleotides, is followed by a slower increase in concentration of the 32 000 polypeptide accompanied by a further increase in purine nucleotide binding. The initial rapid effect of cold stress was mimicked by intravenous infusion of noradrenaline; neither the effect of cold exposure for 24 h nor the effect of intravenous infusion of noradrenaline was prevented by cycloheximide. In contrast, the slow adaptive changes in the mitochondria did not occur in response to prolonged (2 weeks) treatment with noradrenaline, although such treatment did induce the expected tissue hypertrophy accompanied by mitochondrial proliferation. Cold-induced (1 week) increases in purine nucleotide binding and 32 000 polypeptide were not prevented by oxytetracycline. The increase in purine nucleotide binding during the 2nd day of cold exposure was prevented by cycloheximide. The effect of cycloheximide on the increase in the 32 000 polypeptide could not be assessed because sufficiently long-term experiments could not be done with this compound. Thus, the initial response to cold stress appears to involve unmasking of mitochondrial proton conductance pathway sites, most probably mediated by noradrenaline. The slower adaptive response occurs in parallel with tissue hypertrophy, which itself may be mediated by noradrenaline, and appears to require cytosolic but not mitochondrial protein synthesis. However, the changes in mitochondrial composition which result in an increased concentration of proton conductance pathway sites are not mediated by noradrenaline.
Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney
Authors: Upreti R. K.
Published: December 2009

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline–ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.
The stoichiometry and stability of the NADP complexes with manganese(II) ions as studied by electron paramagnetic resonance
Authors: Green M. Kirk , Kotowycz George
Published: December 2009

Magnetic resonance techniques have been applied to study the stability of the complexes formed between Mn(II) ions and NADP in aqueous solutions at a pH of 7.5 and 20 °C. The electron paramagnetic resonance (epr) data indicate that at low Mn(II) ion concentrations ([Mn(II)] < 1 mM; [NADP] ~ 5 mM), a 1:1 complex is formed with an apparent stability constant K1 = 370 ± 50 M−1 at an ionic strength of 0.22 in the presence of 0.20 M Cl−. At high Mn(II) ion concentrations, a Mn(II)2 –NADP species, with an apparent stability constant K2 = 54 ± 17 M−1, is present in significant amounts. When the epr data are corrected for the presence of the MnCl+ ion, the analysis of the new Scatchard plot yields stability constants for the two sites of K1 = 640 ± 90 M−1 and K2 = 88 ± 13 M−1 respectively. The presence of two metal ion binding sites on the NADP molecule has not been observed previously, and previous workers have always analyzed their data in terms of the 1:1 Mn(II)–NADP complex. An epr temperature study of K1 yields a value of ΔH equal to 1.3 ± 0.2 kcal/mol (1 cal = 4.187 J).
Cell-free synthesis of rat liver zinc-thioneins
Authors: Hew Choy L. , Penner Peter E.
Published: December 2009

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea – 50 mM β-mercaptoethanol, by disc gel electrophoresis in 4 M urea – Tris–glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.
The identification of α-adrenergic receptors in adipose tissue by [3H]dihydroergocryptine binding
Authors: Bégin–Heick Nicole , Houlihan Pauleen A. , Sigouin José
Published: December 2009

The 3H-labeHed α-adrenergic antagonist dihydroergocryptine has been found to bind specifically to hamster white adipocyte membranes. Binding is rapid and is inhibited by α but not β antagonists. In addition, adrenergic agonists compete for this binding site in order of affinities equal to their activity as α-adrenergic agonists. The (−) stereoisomer of adrenaline is 10 times more potent than the (+) stereoisomer in inhibiting binding.
Characterization of a stable intermediate in the unfolding of diazoacetylglycine ethyl ester?pepsin by urea
Authors: Ahmad Faizan , McPhie Peter
Published: December 2009

The irreversible unfolding of covalently inhibited swine pepsin by urea was studied by spectrophotometric and viscosity measurements. At pH 4.5 and 25 °C in 8 M urea, a stable intermediate form of the protein was detected. It differed from the native protein by a slight loss of secondary structure and an increased intrinsic viscosity ([η] = 7.5 mL g−1), indicating the intermediate to have an increased molecular volume or to be more asymmetric in shape. The protein was transformed into a random coil form by increases of temperature and pH. Comparison with other results suggested that at pH 6 pepsin is less stable than its inactive precursor, pepsinogen, by about 3 Kcal mol−1 (1 cal = 4.187 J).
The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum
Authors: Glick Bernard R. , Martin William G. , Giroux J. Jean , Williams Ross E.
Published: December 2009

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electrophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 μmol H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 μmol H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction with a polymeric viologen may perform better in a cell-free system aimed at hydrogen production than that from Cl. pasteurianum.
Erratum: The interactions of adenylates with allosteric citrate synthase
Authors: Talgoy Michael M. , Duckworth Harry W.
Published: December 2009

not available
The biosynthesis of catalase
Authors: Ruis Helmut
Published: December 2009

not available
Dopamine-4-O-sulfate: a possible precursor of free norepinephrine
Authors: Buu Nguyen T. , Kuchel Otto
Published: December 2009

Incubation of dopamine-4-O-sulfate with purified bovine dopamine-β-hydroxylase led to the formation of free norepinephrine. The production of free norepinephrine was completely inhibited in the presence of 5.6 mM of fusaric acid, an inhibitor of dopamine-β-hydroxylase. No sulfatase activity was detected in the incubation medium. The reaction of dopamine-4-O- suifate with dopamines-β-hydroxylase followed Michaelis–Menten kinetics, showing an apparent Km of 2.6 mM.