bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and Catalytic Inactivity of the Bacterial Luciferase Neutral Flavin Radical
Authors: KURFÜRST Manfred , GHISLA Sandro , PRESSWOOD Robert , HASTTNGS J. Woodland
Published: March 2005

A luciferase‐bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase‐FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2∼ 20 h at 0°C in air; the Arrhenius δH≠decay being about 170 kJ/mol) and can be prepared in pure form by Sephadex G‐25 chromatography at 0 – 4°C. The pure enzyme‐bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a‐peroxyflavin, the active intermediate in the bioluminescent reaction.
A Multichannel Perifusion System for the Continuous Monitoring of Glycogenolysis and Gluconeogenesis in Isolated Rat Hepatocytes
Authors: CRISP David M. , SORMAN Anna E. , BEIRNE Jennifer M. , ORTON Terry C. , STURDEE Anthony P.
Published: March 2005

A multichannel perifusion system for isolated rat hepatocytes entrapped in a Sephadex matrix is described and criteria for the choice of matrices are discussed. This system overcomes the usual problem of clogged filters and impaired flow rates encountered in suspension perifusion systems, and is assembled from standard widely available components. Gluconeogenic capability and mitochondria1 respiratory control ratios were unaltered. Decreases in trypan blue viability index and respiration rate were small when compared with flask‐incubated hepatocytes. The endogenous rate of glycogenolysis was slightly higher in perifused hepatocytes but hormone response, as judged by glucagon stimulation of glycogenolysis, was unimpaired. The potential of this system is indicated by experiments monitoring glycogenolysis and glucoiieogenesis in recycling and non‐recycling modes.
Effects of Streptozotocin‐Induced Diabetes Mellitus on the Binding and Uptake of Asialoorosomucoïd by Isolated Hepatocytes from Rats
Authors: DODEUR Michèle , DURAND Dominique , DUMONT Jean , DURAND Genévieve , FEGER Jeanne , AGNERAY Jean
Published: March 2005

The binding and the total uptake of [3H]asialoorosomucoíd was studied in normal rats, streptozotocïn‐diabetic rats and insulin‐treated diabetic rats.

The binding of [3H]asialoorosomucoi'd to normal rat hepatocytes gave a curvilinear plot when analysed by the Scatchard method, suggesting either two classes of independent receptor sites with different apparent association constants or a negative cooperativity in binding. The data, plotted according to the method of De Meyts and Roth showed a decrease of the affinity constant with respect to site occupancy.

The Scatchard plot obtained for the [3H]asialoorosomucoid binding to streptozotocin‐diabetic rat hepatocytes was curvilinear. According to the independent site model, the receptor number of each class was decreased, without any change of the apparent association constants. According to the model of De Meyts and Roth, no modification of the interaction factor and apparent affinity constants was observed, whilst the total number of receptor sites was decreased by half in streptozotocin‐diabetic rats as compared with normal rats.

Study of the total uptake of [3H]asialoorosomucoId by hepatocytes from each group of rats showed a decrease by half of the endocytosis in streptozotocin‐diabetic rats as compared with normal rats.

Insulin treatment of the streptozotocin‐diabetic rats restored both parameters studied to normal level, whilst the withdrawal of insulin treatment decreased their level to that of streptozotocin‐diabetic rats.

These results are related to possible modifications of the hepatic binding protein, receptor for the plasma asialoglycoproteins, in the diabetic state.
Sequence Homology between Polyadenylated mRNAs from Transplantable Mouse Tumours of Different Origin, as Compared to mRNAs from Normal Adult or Embryonic Tissues
Authors: MERCIER Gilles , HAREL Jacques
Published: March 2005

Complementary DNA copies (cDNA) were 3H‐labelled and cell‐free‐synthesized on polyadenylated mRNA templates which had been isolated from the cytoplasm of mouse FLS ascites tumour cells. They were hybridized to RNAs from different mouse origins: ascites tumours (FLS, Ehrlich, Krebs 2) passaged in mice, tissue‐cultured Friend erythroleukemia cells, adult liver, adult brain and whole embryos, 10–12 days old. Analysis of the cDNA‐RNA reassociation kinetics showed that the total molecular complexity of FLS cells poly(A)‐rich RNA is equivalent to 11000–12000 different sequences of 1800 residues each, distributed in the three usual classes of high, middle and low abundancies. All or nearly all of these sequences were also found in Ehrlich and Krebs 2 tumour cells with the same frequency distribution as in FLS cells. When comparing the total number and relative abundancies of poly(A)‐rich RNA sequences in ascites tumour cells to other mouse cells, we found slight but reproducible differences for Friend erythroleukemia, more pronounced differences for adult liver or whole embryos, and maximal differences for adult brain. Some of the most abundant poly(A)‐rich RNA species common to all tumour strains either did not exist or were much less abundant in normal tissues. Furthermore, the abundant poly(A)‐rich RNA sequences which are common to ascites tumour cells and fetal tissues were also found with the same frequency distribution in adult liver, where cell proliferation is negligible. This supports the conclusion that most of the frequent poly(A)‐rich sequences, which appeared to be tumour‐specific, cannot simply represent normal mitosis gene transcripts and might in fact be related to the expression of malignancy, at least in old‐established tumour strains of different tissue origins.
Biological Activity and a Partial Amino‐Acid Sequence of Escherichia coli DNA‐Binding Protein I Isolated from Overproducing Cells
Authors: BEYREUTHER Konrad , BERTHOLD‐SCHMIDT Verena , GEIDER Klaus
Published: March 2005

DNA‐binding protein I from Escherichia coli was purified from cells carrying the ssb gene on a multicopy plasmid. In comparison to the strain without the recombinant plasmid the DNA‐binding protein was over‐produced more than 20‐fold. The amount of the protein was measured after the purification steps by gel electrophoresis and radial immunodiffusion. The protein was purified to homogeneity and was active in replication assays like the wild‐type DNA‐binding protein. The assays were enzymatic replication of single‐stranded and double‐stranded fd DNA. E. coli DNA‐binding protein I was further subjected to amino acid sequence analysis. A monomer of the protein consists of 187 residues which correspond to a molecular weight of 19715 with 5% error in analysis. The sequence of the amino‐terminal 40 residues was determined and includes several basic residues of the protein. Sequence comparison between the DNA‐binding protein I from E. coli and that coded by bacteriophage fd reveals similarities suggesting that DNA‐binding protein I may use amino‐terminal residues for binding to DNA like the phage protein.
The Glucose/Glucose‐6‐Phosphate Cycle in the Periportal and Perivenous Zone of Rat Liver
Authors: JUNGERMANN Kurt , HEILBRONN Regine , KATZ Norbert , SASSE Dieter
Published: March 2005

Periportal and perivenous hepatocytes contain different activities (V) of antagonistic key enzymes such as glucokinase and glucose‐6‐phosphatase. In order to get an insight into the metabolism of the periportal and perivenous area the flux rates (v) of the glucose/glucose‐6‐phosphate cycle were calculated on the basis of the Michaelis‐Menten equation using the measured zonal concentrations of glucose and glucose 6‐phosphate, the zonal activities of glucokinase and glucose‐6‐phosphatase previously reported and the half‐saturating substrate con‐ centrations (Km) of the two enzymes found in the literature. The concentrations of glucose were obtained as a first approximation by measuring the concentrations in portal (= periportal) and hepatovenous (= perivenous) blood; those of glucose 6‐phosphate were calculated from the levels determined in microdissected periportal and perivenous liver tissue.

The calculations showed (a) that the overall cycling rates agreed remarkably well with those reported for intact animals and (b) that during a normal feeding rhythm the periportal zone should catalyze net glucose output and the perivenous zone should mediate net glucose uptake, as proposed by the model of ‘metabolic zonation’.
An Extracellular Polysaccharide Produced by Zoogloea ramigera 115
Authors: IKEDA Fumiaki , SHUTO Hidetoshi , SAITO Terumi , FUKUI Tetsuya , TOMITA Kenkichi
Published: March 2005

A weakly acidic polysaccharide was purified from the extracellular zoogloeal ramigera produced by Zoogloeal ramigera 115. The purified polysaccharide was homogeneous as judged by sedimentation analysis, and the average molecular weight was estimated to be about l05 by gel permeation chromatography of the fully methylated preparation. The polysaccharide was composed of d‐glucose, d‐gahCtOSe and pyruvic acid in an approximate molar ratio of 11: 3: 1.5. On the basis of methylation, periodate oxidation, Smith degradation and partial hydrolysis, the following highly branched structure was deduced for the polysaccharide : a long chain mainly consisting of β1→4‐linked glucose residues branching at the C‐3 or C‐6 position of galactose residues which are present in β1→4 or β1→ 3 linkages as the minor component of the long chain; pyruvic acid residues, the sole acidic component, are linked to the nonreducing end and/or 1,3‐linked glucose residues through 4,6‐ketal linkages. The purified polysaccharide was not readily soluble in water and had a high affinity for several metallic ions (e.g. 0.25 pmol Fe3+/mg, and 0.17 μmol Fe2+ mg). Upon addition of metallic ions (1 mM) to a gelatinous aqueous solution of the polysaccharide (K+ form, 0.125 %), more than 80% of it immediately coprecipitated out with them.
Complexing of Glycolipids and Their Transfer between Membranes by the Activator Protein for Degradation of Lysosomal Ganglioside GM2
Authors: CONZELMANN Ernst , BURG Josef , STEPHAN Günther , SANDHOFF Konrad
Published: March 2005

The lysosomal degradation of ganglioside GMZ by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane‐bound ganglioside and water‐soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail.

It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water‐soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome‐bound ganglioside Gm2 is considerably more stable against extraction and degradation than micellar ganglioside.

In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes.

The activator protein is rathcr specific for ganglioside GM2. Other glycolipids (GM3, GM1, GDla and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside G, than ganglioside GM2.
Modified Plasma‐Membrane ATPase in Mutants of Saccharomyces cerevisiae
Authors: ULASZEWSKI Stanislaw , GRENSON Marcelle , GOFFEAU André
Published: March 2005

Mutations affecting the plasma membrane ATPase of Saccharomyces cerevisiae were obtained by selecting mutants resistant to Dio‐9.

In a plasma‐membrane‐enriched fraction of the mutant MG2130, the ATPase activity was resistant to vandate (50% inhibition by 26 μM in the mutant compared to 1.3 μM in the parental strain).

Several catalytic properties of the membrane‐bound ATPase were modified by 60–120% in the mutant which had a higher Km for MgATP and was more heatstable, less sensitive to mercurials, and more stimulated by monovalent cations than the parental type.

A single mutation is responsible for the phenotypes of four independent allelic mutants. Resistance to Dio‐9 in vivo and resistance to vanadate in vitro segregated together in three tetrads issued from a cross between the wild type and mutant. The mutation is semi‐dominant as shown by expression of the mutant phenotype in a heterozygous diploid resulting from the cross between the wild type and mutant.

It is concluded that the pma locus, affected by these mutations, is the structural gene either for the 100000‐Mr subunit of plasma membrane ATPase or for a protein which tightly controls the conformation of the plasma‐membrane ATPase within the membrane.
On the Phosphorylation of Yeast RNA Polymerases A and B
Authors: BRÉNT Bernadette , BUHLER Jean‐Marie , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

In exponentially growing cells, RNA polymerase B is exclusively form BI enzyme with several phosphorylated subunits: B220, B23 and possibly B44.5. In RNA polymerase A an average of fifteen phosphate groups are distributed on the five phosphorylated subunits: A190 (6), A43 (4), A34.5 (2), A23 (1—2) and A19 (1—2). Phosphorylation of enzyme A by a yeast protein kinase in vitro adds less than 1 mol phosphate/mol enzyme but occurs essentially at the physiological sites, as shown by a comparison of the peptide patterns obtained by limited proteolysis of subunits 32P‐labelled in vivo and in vitro. No evidence was found in favour of a modulation of RNA polymerase activity in vitro or in vivo via phosphorylation.
Proteolytic Cleavage of Hansenula anomala Flavocytochrome b2 into Its Two Functional Domains
Authors: GERVAIS Michel , RISLER Yanick , CORAZZIN Sylvie
Published: March 2005

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I.

This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.

Our results are discussed in terms of ‘gene fusion’.
Variation of (2′‐5′)Oligo(adenylate) Synthetase Activity during Rat‐Liver Regeneration
Authors: SMEKENS‐ETIENNE Martine , GOLDSTEIN Jacques , OOMS Henri A. , DUMONT Jacques E.
Published: March 2005

The interferon‐induced double‐stranded RNA‐activated (2′‐5′)oligo(adenylate) synthetase converts ATP into (2′‐5′)oligo(adenylate) [(2′‐5′)oligo(A)] and pyrophosphate. In turn, (2′‐5′)oligo(A) activates a latent endoribonuclease which cleaves single‐stranded RNA.

(2′‐5′)Oligo(A) synthetase activity has been characterized in liver cells of normal and germ‐free rats. This enzyme is predominantly nuclear. After partial hepatectomy, (2′‐5′)oligo(A) synthetase activity decreased rapidly (after 10 h) and markedly to a minimum of 25% of the control after 20 h. This decrease was followed by a slow restoration of the activity. No such change was observed in sham‐operated animals. This important decrease of enzymatic activity occurred during the first few hours of liver regeneration (6–24 h). This period corresponds also to the prereplicative and replicative (18–24 h after hepatectomy) phases of DNA. Detailed kinetics indicated that the loss of (2′‐5′)oligo(A) synthetase activity preceded the onset of the incorporation of tritiated thymidine in DNA and was minimal when the rate of DNA synthesis was maximal. These results and those obtained in culture of cells in vitro are compatible with the hypothesis that the (2′‐5′)oligo(A) system participates in the negative control of cell proliferation.
α1 Acid Glycoprotein: a Small‐Angle Neutron Scattering Study of a Human Plasma Glycoprotein
Authors: LI Zong‐Qi , PERKINS Stephen J. , LOUCHEUX‐LEFEBVRE Marie H.
Published: March 2005

Small‐angle neutron scattering experiments on α1 acid glycoprotein showed that it has a molecular weight of 37000 and a matchpoint of 44.7%2H2O. The molecular weight, the matchpoint and a v̄ of 0.704 ml/g are in agreement with the primary sequence and standard residue volumes for amino acids and carbohydrates.

The radius of gyration RG of α1 acid glycoprotein was found to be independent of concentration in the range 2–11 mg/ml, but increases on going from a buffer containing 0.2 M NaCl to one containing 1 M NaCl. A contrast variation study showed that the RG at infinite contrast is 2.47 nm for the expanded form and 2.19 nm for the contracted form, and that the two Stuhrmann α values are similar at 27 × 10−5. The latter is greater than that expected for globular proteins and are explained by the surface disposition of the five glycan chains on a core of protein in α1 acid glycoprotein. Modelling calculations account for the two RG values in which for the expanded form the glycan chains extend out into the solvent and for the contracted form they either fold back or are splayed out such that they are able to interact with the surface of the protein core.
Two Proteolytic Degradation Products of Calf‐Thymus Poly(ADP‐ribose) Polymerase Are Efficient ADP‐ribose Acceptors
Authors: HOLTLUND Jostein , JEMTLAND Rune , KRISTENSEN Tom
Published: March 2005

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP‐ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP‐ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodcylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112‐kDa polymerase. Isolation of poly(ADP‐ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76‐kDa and 59‐kDa polypeptides in the final polymerase preparation.

When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments.

Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP‐ribosylation sites. Several ADP‐ribosylated fragments were found to be common to all three polypeptides, or to two of them.
Evidence for an Enzyme which Cleaves the Guanidinobenzoate Moiety from Active‐Site Titrants Specifically Designed to Inhibit and Quantify Trypsin
Authors: STEVEN Frank S. , AL‐AHMAD Rajaa K.
Published: March 2005

The serum and ascitic plasma surrounding Ehrlich ascites tumour cells grown in mice contains an enzyme which cleaves 4‐nitropheny‐p‐guanidinobenzoate and 4‐methylumbelliferyl‐p‐guanidinobenzoate. We refer to this enzyme as guanidinobenzoatase. Either of the substrates acts as a competitive substrate for the enzyme when it is assayed with the other substrate. Arginyl‐containing peptide substrates of typsin and thrombin also act as competitive inhibitors of the enzyme but are not cleaved by it. The properties of this enzyme are remarkable in the continuous production of cleavage products from the two substrates, which are both active‐site titrants for trypsin‐like enzymes, rather than stoichiometric cleavage and inhibition of one molecule of enzyme by one molecule of active‐site titrant. The enzyme contains serine in its active centre, being inhibited by both phenylmethylsulphonyl fluoride and diisopropyl‐fluorophosphate.
A Stereochemical and Positional Isotope‐Exchange Study of the Mechanism of Activation of Methionine by Methionyl‐tRNA Synthetase from Escherichia coli
Authors: LOWE Gordon , SPROAT Brian S. , TANSLEY Gaynor
Published: March 2005

Methionyl‐tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5′‐[(R)‐α17O]triphosphate with inversion of configuration at Pα. Furthermore methionyl‐tRNA synthetase does not catalyse positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl‐enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl‐tRNA synthetase catalyses the activation of methionine by an associative ‘in‐line’ nucleotidyl transfer mechanism.

A kinetic study of positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the presence of methionine, Mg2+ and methionyl‐tRNA synthetase showed that torsional equilibration (18O exchange into the Pα—O—Pβbridge) occurs faster than tumbling (18O exchange into P7 by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E · Met‐AMP · Mg[18O2]PPi complex.
Enzymic Properties of an N‐Acetylglucosaminide 3‐α‐l‐Fucosyltransferase of a Wheat‐Germ Agglutinin‐Resistant Melanoma Clone
Authors: PRIEELS Jean‐Paul , MONNOM Dominique , PERRAUDIN Jean‐Paul , FINNE Jukka , BURGER Max
Published: March 2005

A fucosyltransferase was solubilized by extraction with Triton CF‐54 from a wheat‐germ agglutinin‐resistant variant of mouse B16 melanoma. Through affinity chromatography on GDPhexanolamine–Sepharose a 44‐fold enrichment of its specific activity was obtained. Analysis of its specificity indicated that the enzyme is an N‐acetylglucosaminide 3‐α‐l‐fucosyltransferase, which is able to transfer fucose to oligosaccharides containing Gal(β‐4)GlcNAc and Gal(β‐4)Glc structures. The enzyme is activated by divalent cations and has a maximum of activity at pH 5. It is unable to transfer fucose to sialylated glycoproteins, 6‐α‐sialyllactose or 3‐α‐sialyllactose. As suggested by its precipitation in the presence of antibodies raised in rabbit against a soluble human milk N‐acetylglucosaminide 3‐α‐l‐fucosyltransferase, these two enzymes seem to be structurally related.
Coordinate and Non‐coordinate Estrogen‐Induced Expression of A and B Groups of Vitellogenin Genes in Male and Female Xenopus Hepatocytes in Culture
Authors: WOLFFE Alan P. , TATA Jamshed R.
Published: March 2005

The concentration of mRNA transcribed from A and B groups of vitellogenin genes, induced by estrogen added to primary cultures of hepatocytes from male and female adult Xenopus, was measured by a technique of filter disc hybridization with cloned Xenopus vitellogenin cDNA probes.

In cells from naive male Xenopus (i.e. not previously exposed to hormone in vivo or in vitro), the two groups of the multigene family were non‐coordinately expressed during the early stages of response to estradiol in vitro. Only B group transcripts could be detected for the first 2–3 h. At later times, or upon successive additions of estradiol to the male cell cultures, both A and B group mRNAs accumulated at the same rate and to the same extent. In female hepatocytes both groups of mRNAs accumulated in parallel at all stages of their response to estrogen, reaching levels 10‐fold higher than in naive male cells similarly exposed to the hormone. However, hepatocytes from male Xenopus that had received a single injection of estradiol 5 weeks before the cells were prepared, now exhibited identical rates and extent of accumulation of A and B groups of vitellogenin mRNAs to those observed in female cells.

Pulse‐labeling of RNA in cultured male Xenopus hepatocytes confirmed that the coordinate and non‐coordinate accumulation of mRNAs were largely a function of differential or equal transcription of the A and B groups of vitellogenin genes. A phenomenon analogous to the hormone‐induced shift from non‐coordinate to coordinate expression of two groups of genes of the same multigene family has not been described previously, and we discuss possible mechanisms underlying the transition.
6‐Phospho‐d‐gluconate Dehydrogenase from Pseudomonas fluorescens
Authors: STOURNARAS Christos , MAURER Peter , KURZ Gerhart
Published: March 2005

The 6‐phospho‐d‐gluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) from Pseudomonas fluorescens, a B‐side stereospecific enzyme, is active with both NAD+ and NADP+, having a specific activity of the homogeneous enzyme of 121 μmol NADH and 23 μmol NADPH, respectively, formed min−1 mg protein−1. The pI of the native enzyme is 4.62, the pH optimum is about 8.2.

The molecular weight of the native enzyme has been determined to be 126000 by sedimentation equilibrium studies. The molecular weight of the polypeptide chains composing the enzyme has been found to be 32000 bydodecylsulfate/polyacrylamide gel electrophoresis and 31000 by sedimentation equilibrium studies in presence of 6 M guanidine hydrochloride. The native enzyme is composed of four polypeptide chains.

Reacting enzyme centrifugation studies gave at pH 8.2 a sedimentation coefficient s20, w of 8.04 S and a diffusion coefficient D20, w of 6.56 F, resulting in a molecular weight of 115000 for the catalytically active form. Thus, the enzyme is active as the tetramer. So far the enzyme from P. fluorescens is the sole 6‐phospho‐d‐gluconate dehydrogenase (decarboxylating) composed of four polypeptide chains.
The Major Proteins from HeLa Cells
Authors: LENSTRA Johannes A. , BLOEMENDAL Hans
Published: March 2005

Subcellular fractionation allows a classification of the most abundant proteins from HeLa cells, detected after two‐dimensional gel electrophoresis. The positions of the spots belonging to actin, α‐tubulin, β‐tubulin and vimentin were indentified on the basis of literature data. Most other relatively abundant proteins could be identified, i.e. the cytokeratins, membrane proteins, elongation and initiation factors of protein biosynthesis.

From the latter group, the elongation factors eEF‐Tu and EF‐2 and the initiation factor eIF‐4A were identified separately.

The intensity of the spot corresponding to eEF‐Tu, described as one of the most abundant proteins in all eucaryotic cells [L. I. Slobin (1980) Eur. J. Biochem. 110, 555–563], was affected strongly by aggregation during isoelectric focusing. A method is described for extracting this protein from cultured cells by using its particular aggregation properties.
Primary Structure of Histone H2A from Gonads of the Starfish Asterias rubens
Authors: MARTINAGE Arlette , BELAICHE Denise , DUPRESSOIR Thierry , SAUTIERE Pierre
Published: March 2005

The complete amino acid sequence (124 residues) of histone H2A from gonads of the starfish Asterias rubens has been established from automated sequence analyses of large fragments obtained by staphylococcal protease digestion of histone H2A and by limited hydrolysis of H2A‐H2B complex with mouse submaxillary gland protease and from structural studies of peptides generated by enzymatic hydrolyses of these fragments or of the protein.

By comparison with calf homologus histone, the starfish histone H2A shows 5 deletions and 12 substitutions. Half of the substitutions are non‐conservative.

Microheterogeneities were found at positions 18, 40 and 50 and result in the existence of at least two variants of starfish gonad histone H2A.
Secondary Structure of Chondroitin Sulphate in Dimethyl Sulphoxide
Authors: SCOTT John E. , HEATLEY Frank , JONES Malcolm N. , WILKINSON Allan , OLAVESEN Anthony H.
Published: March 2005

1H NMR spectra were obtained in fully deuterated dimethyl sulphoxide of sodium chondroitin 4‐sulphate and of the tetrasaccharide, hexasaccharide and octasaccharide prepared from it. Two types of NH resonance were observed, at 7.6 ppm and 8.8 ppm. The latter was the only one in the polymer spectrum, whereas both were present in the oligomer spectra: N‐acetylchondrosinate produced only the 7.6‐ppm signal. The upfield resonance (7.6 ppm) is characteristic of ‘normal’, monomeric acetamido sugars and disaccharides, while the downfield resonance (8.8 ppm) suggests a hydrogen‐bonded NH. The elongation of disaccharide to tetrasaccharide produces a new environment, involving a hydrogen bond at the non‐reducing disaccharide NH. A structure, with three hydrogen bonds per tetrasaccharide unit, is proposed which accounts for the quantitative and qualitative aspects of the NMR spectra of polymers and oligomers. A laser light‐scattering study showed that sodium chondroitin 4‐sulphate has the same molecular weight in water as in dimethyl sulphoxide. The observed hydrogen bonds are therefore intramolecular, as required by the proposed structure, and probably exist in water as well as in dimethyl sulphoxide.
Sequential Individual Resonance Assignments in the 1H Nuclear‐Magnetic‐Resonance Spectrum of Cardiotoxin VII 2 from Naja mossambica mossambica
Authors: HOSUR Ramakrishna V. , WIDER Gerhard , WÜTHRICH Kurt
Published: March 2005

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of cardiotoxin VII2 from Naja mossambica mossambica is described and documented. The assignments are based entirely on the amino acid sequence and on two‐dimensional NMR experiments at 500 MHz. Individual assignments were obtained at 45 C for the backbone protons of 56 out of the total of 60 amino acid residues, the exceptions being the N‐terminal dipeptide segment Leu‐1–Lys‐2–, Pro‐8 and Pro‐15. Complete assignments of the non‐labile hydrogen atoms of the side chains were obtained for 37 residues, and for Asn‐4 and Asn‐19 the δ amide protons were also identified. For 19 long side chains the individual assignments include only the backbone and C‐β proton resonances; these are Gln‐5, Pro‐9, Pro‐33, Pro‐43, Leu‐47, all three methionines, two arginines and nine lysines. The chemical shifts for the assigned resonances at 45°C are listed for an aqueous solution at pH 3.6. A preliminary interpretation of the sequential connectivity patterns indicates that approximately 30 out of the total of 60 amino acid residues in cardiotoxin VII2 are in extended, β‐type secondary structures, and there is no indication for the formation of α‐helical structure.
The Proof by 13C‐NMR Spectroscopy of the Predominance of the C5 Pathway over the Shemin Pathway in Chlorophyll Biosynthesis in Higher Plants and of the Formation of the Methyl Ester Group of Chlorophyll from Glycine
Authors: PORRA Robert J. , KLEIN Otto , WRIGHT Peter E.
Published: March 2005

The 13C‐NMR spectrum of chlorophyll a formed when excised etiolated maize leaves were greened in the presence of [1‐13C]glutamate showed that the four methine bridge carbons (C‐5, C‐10, C‐15 and C‐20) and four pyrrole α‐carbons (C‐4, C‐9, C‐14 and C‐16) were considerably enriched relative to the natural‐abundance spectrum thus providing unequivocal proof of the operation of the C5 pathway of 5‐aminolaevulinate formation during chlorophyll biosynthesis in maize. [The carbon numbering system used is that recommended by IUB, see Pure Appl. Chem. 51, 2251–2304 (1979).]

The 13C‐NMR spectrum of chlorophyll a formed during greening in the presence of [2‐13C]glycine showed that little or no enrichment of the above‐mentioned methine bridge or pyrrole α‐carbons had occurred thus indicating that the Shemin pathway makes little or no contribution to chlorophyll formation in greening maize. There was, however, considerable enrichment of the methoxyl carbon of the methoxycarbonyl group attached to isocyclic ring carbon C‐132. This shows that the C‐2 carbon of glycine is used to label the C1 pool and subsequently the methyl ester carbon of magnesium‐protoporphyrin IX‐monomethyl ester which is an intermediate in chlorophyll biosynthesis.

The 13C‐NMR spectrum of chlorophyll a obtained when greening occurs in the presence of [5‐13C]glutamate showed considerable enrichment of the two carboxyl carbons of chlorophyll a (C‐133 and C‐173). This spectrum also showed that enrichment, equivalent to 15% of that occurring at the carboxyl carbons, occurred at the four methine bridge carbons and four relevant pyrrole α‐carbons. This is consistent with the conversion of [5‐13C]glutamate to [1‐13C]2‐oxoglutarate (or glutamate) via [5‐13C]2‐oxoglutarate and recycling in the tricarboxylic acid cycle: the soformed 1‐13C‐labelled substrate would then be incorporated into chlorophyll by the C5 pathway. This assessment of the amount of [5‐13C]glutamate incorporated into chlorophyll after recycling as 2‐oxoglutarate in the tricarboxylic acid cycle is used not only to confirm the view that the C5 pathway is the major, possibly sole, route for chlorophyll biosynthesis in higher plants but is also used to negate previous assertions of the involvement of the Shemin pathway based on comparison of the incorporation of 2‐oxoglutarate 14C‐labelled at C‐1 or C‐5.

In these studies we also found that chlorophyll a purified by silica‐gel thin‐layer chromatography contained allomerization products and was unsuitable for 13C‐NMR spectroscopy.
The Fate of Xenopus and Locust Vitellogenins Made in Xenopus Oocytes
Authors: LANE Charles D. , CHAMPION Janet , COLMAN Alan , JAMES Tharappel C. , APPLEBAUM Shalom W.
Published: March 2005

Xenopus liver or locust fat body RNA injected into Xenopus oocytes directs the formation of a high‐molecular‐weight species which is found transiently within the oocyte in a membranous vesicle fraction. The conversion of such internally generated Xenopus vitellogenin to yolk platelet proteins is reduced by the presence in the incubation medium of anti‐vitellogenin antibodies, or of tunicamycin, or of a mixture of colchicine and cytocholasin. but neither export nor conversion is blocked by removal of the follicular layers which surround the oocyte. Moreover, surrounding mRNA injected defolliculated oocytes with an excess of small defolliculated oocytes leads to a reduction in lipovitellin accumulation in the cells making vitellogenin and the appearance of lipovitellin in the platelets of the uninjected feeder cells.

We propose that newly‐made locust and Xenopus vitellogenins are sequestered in vesicles and are then secreted by the oocyte, the locust species being further modified by cleavage just before export. Thus processing of heterologous yolk precursors follows the pathway characteristic of the cell type used to prepare the donor RNA. We suggest that the import mechanism discriminates between the exported locust and frog proteins, and that only the Xenopus vitellogenin subsequently enters the oocyte, where it is converted to lipovitellin and phosvitin by an enzyme present in the yolk platelets. Thus we explain the apparently paradoxical observations that internally generated and externally supplied frog vitellogenin are both converted to yolk platelet proteins, whilst injected vitellogenin is not.
A Novel One‐Carbon Carrier (Carboxy‐5,6,7,8‐tetrahydromethanopterin) Isolated from Methanobacterium thermoautotrophicum, and Derived from Methanopterin
Authors: KELTJENS Jan T. , DANIELS Lacy , JANNSEN Henny G. , BORM Paul J. , VOGELS Godfried D.
Published: March 2005

During short‐term labeling experiments, cells of Methanobacterium thermoautotrophicum incorporated a substantial part of 14CO2 in a compound with a bright yellow fluorescence on dry thin‐layer chromatography plates and called yellow fluorescent compound (YEC) [Daniels, L. and Zeikus, J. G. (1978) J. Bacteriol. 136, 75–84]. This compound was extracted and purified by ion‐exchange column chromatography with formic acid gradients up to 0.3 M. Out of 325 g wet cells of M. thermoautotrophicum about 4 mg of the compound were isolated. This material and some degration products obtained from it were studied by means of chemical decomposition, ultraviolet‐visible‐light spectroscopy and preliminary 1H‐NMR spectroscopy. It has structural elements in common with methanopterin (see preceding paper in this journal); these elements are a pterin group, glutamate, a hexosamine.

The pterin in this compound is present in reduced form, presumably as 5,6,7,8‐tetrahydromethanopterin, and the additional one‐carbon unit is probably present as a carboxy group.

Probably the first step of methanogenesis implies a carboxylation of methanopterin and a concomitant reduction of the pterin. The trivial name carboxy‐5,6,7,8‐tetrahydromethanopterin is introduced for the compound.
Investigation of the Primary Photochemistry of Bacteriorhodopsin by Low‐Temperature Fourier‐Transform Infrared Spectroscopy
Authors: SIEBERT Friedrich , MÄNTELE Werner
Published: March 2005

The method of Fourrier‐transform infrared difference spectroscopy was applied to investigate the transition at 77K of bacteriorhodopsin in its light‐adapted form to K610, the first intermediate which is stable at low temperature. In addition to unmodified bacteriorhodopsin, bacteriorhodopsin in 2H2O and bacteriorhodopsin containing [15‐2H]retinal was used. The results show that major rearrangements occur in the Schiff base in this transition. It is not possible to identify a C=N stretching vibration of the Schiff base in K610. The identification of an N‐H bending vibration in K610 shows that the nitrogen of the previous Schiff base still has a proton attached. The fingerprint region exhibits very unusual features for K610 and bears no similarity to protonated retinylidene Schiff base model compounds of any isomeric composition. Therefore, no conclusions on the isomeric state of the retinal in K610 can be drawn. The spectra show that the terminal part of the retinal is predominantly reflected in the difference spectra. This indicates that the most polar part of the retinal is located near the Schiff base. We have evidence for protein molecular changes occurring in this transition at 77K.
Characterization of Phosphorylated and Native cGMP‐Dependent Protein Kinase
Authors: HOFMANN Franz , FLOCKERZI Veit
Published: March 2005

Pure preparations of cGMP‐dependent protein kinase are autophosphorylated in the presence of cAMP and cGMP. The stoichiometry and functional significance of this reaction has been studied. Pure preparations of cGMP‐dependent protein kinase contained 1.4 ± 0.1 (7) mol phosphate/mol subunit as determined by chemical phosphate analysis. The protein‐bound phosphate was hydrolyzed by 1 M NaOH, but was stable in 0.1 M HCl or 0.8 M NH4OH or hot 16% trichloracetic acid. In the presence of cAMP and [γ‐32P]ATP. Mg another 2.5 ± 0.2 (5) mol phosphate/mol subunit were incorporated into the enzyme yielding a total of 4.2 ± 0.06 (5) mol phosphate/mol subunit. cGMP also stimulated the phosphorylation of the enzyme although to a lower extent than cAMP. The autophosphorylation reaction was half‐maximally stimulated at 0.12 μM and 7.0 μM cGMP and cAMP, respectively. cAMP‐dependent autophosphorylation of the native enzyme did not change the apparent KA for cGMP in a phosphotransferase assay (KA= 0.15 μM) but decreased the KA for cAMP from 19.6 μM to 1.75 μM. Phosphorylation did not affect the apparent Km for the substrate peptide but doubled the apparent V of the phosphotransferase reaction determined in the presence of saturating concentrations of cGMP or cAMP. Phosphorylation did not change the affinity of the enzyme for cGMP, when cGMP binding was determined in the absence of ATP (apparent Kd= 18 nM). ATP · Mg decreased the affinity of the native enzyme for cGMP about threefold to an apparent Kd of 54 nM; in contrast, ATP · Mg had only a minimal effect on the binding kinetics of the phosphorylated enzyme. These results support the notion that autophosphorylation of cGMP‐dependent protein kinase considerably influences the kinetic parameter of the enzyme.
Interaction of Dicyclohexylcarbodiimide with the Proton‐Conducting Pathway of Mitochondrial H+‐ATPase
Authors: KOPECKY Jan , GUERRIERI Ferruccio , PAPA Sergio
Published: March 2005

In this paper a study is presented of the interaction of N,N′‐dicyclohexylcarbodiimide (DCCD) with the H+‐ATPase of mitochondria. Inhibition of proton conduction by the membrane sector (Fo) of the H+‐ATPase has been characterized by kinetics analysis of the effect of DCCD on the anaerobic decay of ΔμH+ set up by respiration in submitochondrial particles containing (ESMP) or deprived (USMP) of the catalytic sector (F1).

The inhibitory effect of DCCD was directly related to the amount of DCCD bound independently of the time of incubation and of the concentration applied. Both in ESMP or USMP 2—3 nmol DCCD/mol Fo were required for the maximal inhibition of proton conduction.

In ESMP the anaerobic relaxation of ΔμH+ is biphasic. Both phases were inhibited by DCCD and the titration curve indicated that the slow phase is directly related to the hydrolytic activity of H+‐ATPase. In USMP the anaerobic relaxation of ΔμH+ is monophasic. DCCD, at low concentrations, restored the biphasic pattern with the appearance of a transient fast phase having a kinetic constant higher than that of the control. Whilst the slow phase was immediately inhibited by DCCD the fast phase started to be inhibited only at concentrations higher than 1 nmol DCCD bound/mg protein.

The data reported support the view that the fast phase of proton conduction reflects a conformational state of Fo, with higher proton conductivity, which is induced by high aerobic ΔμH+.

Electrophoretic analysis of the labeling by [14C]DCCD of membrane polypeptides indicated that the inhibition of the state of low proton conductivity of Fo can be correlated with binding to the 8000‐Mr form of DCCD‐binding proteolipid, whilst inhibition of the state of high proton conductivity of Fo can be correlated with binding to the 16000‐Mr polypeptide.
A Calcium‐Activated Protease which Preferentially Degrades the 160‐kDa Component of the Neurofilament Triplet
Authors: ISHIZAKI Yasuki , TASHIRO Tomoko , KUROKAWA Masanori
Published: March 2005

A calcium‐dependent protease fully active with 0.2 mM Ca2+ was found associated with the neurofilament enriched cytoskeleton of the rat spinal cord prepared by the treatment with Triton X‐100. The enzyme preferentially degrades the 160‐kDa component of the neurofilament triplet. In addition, a soluble calcium‐dependent protease activity was found in the supernatant from the spinal cord, which degraded a variety of cytoskeletal proteins including the neurofilament triplet, glial fibriallary acidic (GFA) protein, actin, tubulin, and a high molecular weight protein associated with microtubules. The possibility that the cytoskeleton‐bound activity is an artefectual association of the soluble enzyme to the cytoskeleton seems to be negated on the basis of the following dissociation and reassociation experiments. The protease activity remained associated with the cytoskeleton in the physiological ionic strength, and was not completely dissociated from it until the KCI concentration was raised to 0.6 M. When the 0.6 M KCI‐extract was dialysed against salt‐free buffer to remove KCI, and added back to the protease‐free cytoskeletal pellet, proteolytic activity was partially restored. Full activity returned only when the extract and the protease‐free cytoskeletal pellet were first combined in the presence of 0.6 M KCI, and then slowly reassociated by dialysis against salt‐free buffer. Dissociated enzyme was rapidly inactivated at 37°C in the presence of Ca2+. These results suggest the structural association of the protease with the cytoskeleton under the physiological condition.
Mitochondrial Pantetheinephosphate Adenylyltransferase and Dephospho‐CoA Kinase
Authors: SKREDE Sverre , HALVORSEN Ola
Published: March 2005

Pantetheinephosphate adenylyltransferase and dephospho‐CoA kinase, which are last in the sequence leading to the biosynthesis of CoA, are localized in mitochondria (about 30% of the activity in the cell) and cytosol. The first three enzymes in the pathway are only found in cytosol [Skrede, S. and Halvorsen, O. (1979) Biochem. Biophys. Res. Commun. 91, 1536–1542].

Both enzymes are present in the mitochondrial matrix, but dephospho‐CoA kinase also has a more ‘external’ pool and is less easily removed from the membrane fractions of the mitochondria.

In accordance with the submitochondrial fractionation studies, pantetheinephosphate adenylyltransferase is to a larger extent activated by detergents or mechanical disruption of the mitochondria than is dephospho‐CoA kinase.

In intact mitochondria, the biosynthesis of CoA from 4‐phosphopantetheine was stimulated by uncoupling agents, probably because of facilitated penetration of the substrate through the inner mitochondrial membrane and or of leakage of a regulator.

Intact mitochondria could increase the synthesis of CoA 5–10 times, when conditions were chosen to promote biosynthesis from 4‐phosphopantetheine and to remove CoA by acylation and/or ‘leakage’ out of the mitochondria.

The kinetic properties of both mitochondrial enzymes are quite similar to the cytosolic ones (but our reinvestigation of the latter enzymes only partially confirm previous studies). Km for 4‐phosphopantetheine and dephosplio‐CoA were 0.015 and 0.01 mmol/1, respectively, for the mitochondrial enzymes.

Mitochondrial biosynthesis of CoA was strongly inhibited by dephospho‐CoA at the pantetheinephosphate adenylyltransferase level and more slightly by CoA at the dephospho‐CoA kinase level
Kinetic Properties of Hxdrogenase Isolated from Desulfovibrio vulgaris (Hildenborough)
Authors: GRANDE Hans J. , van BERKEL‐ARTS Anita , BREGH Jan , van DIJK Kees , VEEGER Cees
Published: March 2005

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artifical donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n= 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.
Structural and Functional Properties of Cytochrome c Oxidase from Bacillus subtilis W23
Authors: DE VRIJ Wim , AZZI Angelo , KONINGS Wil N.
Published: March 2005

The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkylglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57000, 37000 and 21000. This is the first report on a bacterial aa3‐type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidase. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.