bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of a Specific Histone Hl Protein Kinase from Mouse Plasmacytoma
Authors: QUIRIN‐STRICKER Christiane , SCHMITT Michèle
Published: March 2005

A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high‐salt wash, ammonium sulfate precipitation, chromatography on DEAF‐cellulose, hydroxyapatite and Sephadex G‐200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2‐mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.4‐7.8. After purification, the histone H1 kinase was not stimulated by cAMP or cGMP. It was not inhibited by the heat. cAMP‐dependent protein kinase inhibitor from beef heart. It utilized preferentially GTP over ATP as phosphate donor. Km values for ATP and GTP were 58 μM and 1. 4μM respectively; the Km for histone H1 was 14 μg ml−1. The molecular weight was approximately 90000 by gel‐exclusion chromatography. Analysis of the purified H1‐specific protein kinase by polyacrylamide gel electrophoresis in dodecylsulfate showed two bands having molecular weights of approximately 64000 and 54000. Many characteristics of this kinase were similarto those of the chromatin‐bound protein kinase reported by other workers in rapidly proliferating cells.
Specificity of a Serum Peptidase Hydrolyzing Thyroliberin at the Pyroglutamyl‐Histidine Bond
Authors: BAUER Karl , NOWAK Peter , KLEINKAUF Horst
Published: March 2005

The substrate specificity of a serum enzyme which degrades thyroliberin (< Glu‐His‐Pro‐NH2) into pyroglutamic acid and His‐Pro‐NH2 has been investigated and compared with that of the pyroglutamyl aminopeptidase from calf liver. The latter enzyme has a broad specificity, causing rapid degradation of thyroliberin, pyroglutamyl β‐naphthylamide and luliberin. In contrast, the serum enzyme causes rapid stereospecific cleavage only of the pyroglutamyl‐histidine bond of thyroliberin and closely related peptides. Compounds such as < Glu‐Ala, < Glu‐His and pyroglutamyl β‐naphthylamide, which are known substrates of the pyroglutamyl atninopeptidases (such as the liver enzyme), are not substrates of the serum enzyme, and inhibit it only poorly. Pyroglutamyl‐containing peptides such as luliberin and neurotensin and thyroliberin analogues such as lld‐thyroliberin, < Glu‐His‐Pro‐NHCH3, < Glu‐His‐Pro‐Gly‐NH2 and < Glu‐Phe‐Pro‐NH2 inhibit effectively the degradation of thyroliberin by the serum enzyme, but are not hydrolyzed by this enzyme. The high specificity of the serum enzyme implies a physiological function.
Synthesis and Posttranslational Segregation of Glyoxysomal Isocitrate Lyase from Castor bean Endosperm
Authors: ROBERTS Lynne M. , LORD J. Michael
Published: March 2005

Total Polyadenylated RNA isolated from 3‐day‐old germinating caster bean endosperm was translated in the rabbit reticulocyte lysate cell‐free protein‐synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase synthesized in vivo had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several protein encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not contranslationally segregated into the lumen of the microsomal vesicles.

Intact 3‐day‐old endosperm tissue was labeled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes.

The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.
Binding of tRNA to Escherichia coli Ribosomes as Measured by Velocity Sedimentation
Authors: SCHMITT Marion , MÖLLER Achim , RIESNER Detlev , GASSEN Hans Günter
Published: March 2005

We followed the binding of initiator and elongator tRNA to 70‐S ribosomes and its subunits by velocity sedimentation in the analytical ultracentrifuge. This technique shows the advantage over the previously used methods (adsorption of the complexes to nitrocellulose filters or fluorescence titrations) in that no kinetic effects obscure the equilibrium data and that none of the components has to be chemically modified. The concentrations of the macromolecular compounds are kept constant and the binding equilibria are shifted by varying the Mg2+ concentration in a range which is accessible to experimental analysis.

Free 30‐S ribosomes bind no tRNA, whereas one tRNA molecule is bound to 50‐S ribosomal subunits. In the presence of the cognate condon one tRNA can be associated with the small subunit. Free, programmed, or misprogrammed 70‐S ribosomes bind exactly two elongator tRNAs. Only the initiator tRNA does discriminate significantly between the two ribosomal sites when bound to a ribosome. A‐U‐G complex.
Disulphide Exchange Reactions in the Control of Enzymic Activity
Authors: STEVEN Frank S. , GRIFFIN Margaret M. , SMITH Ronald H.
Published: March 2005

The causative agent in kale poisoning of cattle is the disulphide, dimethyl disulphide. Typical signs of kale poisoning are circulating dimethyl disulphide in the blood, Heinz body (deposits of methaemoglobin) formation in the erythrocytes and loss of body weight. Experiments in vivo have demonstrated that dimethyl disulphide can participate in disulphide exchange reactions which have a controlling role on proteolytic enzymes. We chose the enzyme trypsin, the active centre of which is maintained by a significant disulphide bond and the enzyme papain which is a thiol protease, to illustrate the mechanisms involved in these disulphide exchange reactions.
Exclusive Localization of Colicin A in Cell Cytoplasm of Producing Bacteria
Authors: CAVARD Danièle , BERNADAC Alain , LAZDUNSKI Claude
Published: March 2005

The production of colicin A in Citrobacter freundii and in Escherichia coli was studied. After induction with low concentrations of mitomycin C, these organisms differed with regards to cell growth, cell viability, and kinetics of colicin A biosynthesis. Despite these differences, immunoferritin labeling on ultra‐thin sections of induced frozen cells demonstrated that colicin A was located exclusively within the cell cytoplasm in both types of bacteria. By using protein markers, it was shown that at no times after induction was colicin A accumulated in the periplasmic space or in inner or outer membranes. These results were confirmed by a biochemical approach. For at least 3 h after induction, colicin A remained associated with producing cells and no colicin A activity was found in the periplasmic space. These results are discussed with reference to the synthesis and export of other bacteriocins.
Isoleucyl‐tRNA Synthetase from Baker's Yeast
Authors: FREIST Wolfgang , VON DER HAHR Frsiedrich , CRAMER Friedrich
Published: March 2005

The order of substrate addition to isoleucyl‐tRNA synthetase from baker's yeast has been investigated by steady‐state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncopetitively with respect to ATP, namely purineriboside (=nbularin), 3′‐deoxy‐adenosine (=cordycepin), 8‐amino‐adenosine and 8‐azido‐adenosine 5′‐triphosphates. The inhibition studies were done in the amino‐acylation and in the pyrophosphate exchange reaction, the aminoacylation eas investihated in the absence and presence of inorganic pyrophosphatase. Additionally bisubstrate kinetics and product inhibition studies were carried out.

The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. Yhe results of the pyrophosphate exchange studies are consistent with formation of E. Ile‐AMP‐ATP.Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and iso‐leucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter‐ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi‐uni uni‐bi ping‐pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter‐ter mechanism.
Hyperbolic Kinetics of the Electrophoretic Carrier of Ca2+ Uptake in Liver Mitochondria
Authors: AFFOLTER Hubert , CARAFOLI Ernesto
Published: March 2005

The use of a fast‐responding Ca2+‐selective electrode has allowed the simultaneous measurement of the true Ca2+‐activity in the medium and of the Ca2+‐transport rate, eliminating the uncertainties connected with the use of indirect methods of estimating Ca2+ transport rates (e.g. O2‐stimulation, K+‐efflux). The results on the kinetics of Ca2+ uptake by rat liver mitochondria obtained in this study are qualitatively similar to those obtained by Bragadin et al. [Bragadin,M.,Pozzan, T, & Azzone, G. F. (1979) Biochemistry, 18, 5973‐5978]by monitoring the K+‐efflux from mitochondria. In the valinomycin‐driven Ca2+ uptake, the Ca2+ translocase is the limiting factor, whereas in the system driven by succinate extraneous limiting factors play a role. Thus, liver mitochondria, when studied under appropriate non‐limiting conditions, exhibit a hyperbolic dependence of the Ca2+ uptake rate on Ca2+‐activity analogous to that of mitochondria from other tissues. The sigmoidal dependence of the uptake rate on Ca2+‐activity in liver mitochondria, observed under conditions which produce hyperbolic kinetics in mitochondria from other tissues, can thus be attributed to factors which are extraneous to the Ca2+ transport system per se.
Primary Structure of a Protamine Isolated from the Sperm Nuclei of the Dog‐Fish Scylliorhinus caniculus
Authors: SAUTIÈRE Pierre , BRIAND Gilbert , GUSSE Michel , CHEVAILLIER Philippppe
Published: March 2005

A protamine was isolated from muture sperm nuclei of the dog‐fish Scylliorhinum caniculus. It contains 31 amino acids per molecule and only five types of residues: arginine (20), glycine (6), serine (3), alanine (1) and tyrosine (1). The primary structure of this protamine is reported. The N‐terminal sequence contains the four hydroxylated amino acids of the molecule; the C‐terminal region shows a sequence of eleven adjacent residues of arginine and contains all the glycine residues present in the protein. The structure of this scylliorhinine is compared to the amino acid sequence of other sperm protamines whose structure has been previously published. The presence of a modified tyrosine residue in some preparation is discussed in relation to sperm maturation.
Affinity Laeling of Horse‐Liver Alcohol Dehydrogenase by 3‐Chloroacetylpyridine – Adenine Dinucleotide
Authors: FOUCAUD Bernard , BIELLMANN Jean‐Franscois
Published: March 2005

3‐Chloroacetyl‐pyridine‐adenine dinuleotide (cla3PdAD+) is a reative NAD+ analogue with the highest possible conservation of the coenzyme structure on its pyridine moiety; it has been designed to alkylate an enzyme catalytic group from the active site. It interactions with the horse liver alcohol dehydrogenase as well as the interaction of structurally related compounds with this enzyme have been investigated for their specificity and their selectivity. The following results have been obtained. (1) clac3PdAD+ is active as coenzyme and its isosteric analogue 3‐propionylpyridine‐adenine dinucleotide (pp3PdAD+) does not display cooperative properties in its activity and binding with the horse liver alcohol dehydrogenase, (2) The horse liver alcohol dehydrogenase is irreversily inactivated by clac3PdAD+ subsequently to the formation of a specific reversible binarycomplex, with a Kd of 11 ± 3 μM: NAD+ protects the enzyme in a strictly competitive way, with a Kd of 67μM at pH 7.5, 22°C. No inactivation occurs with 3‐chloroacetylpyridine and/with 4‐chloroacetylpyridine‐adenine dinucleotide (clac4PdAD+). (3)The pH dependence of the rate of the enzyme inactivation by clac3PdAD+ reflects the variation with pH of the dissociation constant of the coenzyme analogue; this variation is similar to that of NAD+. (4) The formation of ternary complexes with ethanol and pyrazole increases the rate of enzyme inactivation, consistently with the enzyme mechanism. From this effect, their dissociation constant could e calcualted and compared to literature data related to other ternary complexes. In contrast, imidazole is shown to decrease the interaction between clac3PdAD+ and the horse liver alcohol dehydrogenase. (5)The inactivation of the enzyme biphasic at high clac3PAD+ concentration, and this iphasicity does not result from a heterogeneity of the preparation of the enzyme or coenzyme analogue. Similarities between this biphasicity and a substrate‐induced biphasicity under certain conditions of inactivation of the enzyme are discussed. It is concluded that clac3PdAD+ is highly specific, and a valuable tool for structural and mechanistic studies of the horse liver alcohol dehydrogenase.
Up‐Regulation in Vascular Endothelial Cells of Binding Sites of High Density Lipoppppprotein Induced by 25‐Hydroxycholesterol
Authors: TAUBER Jean‐Pierre , GOLDMINZ David , GOSPODAROWICZ Denis
Published: March 2005

Exposure of bovine vascular endothelial cell cultures to 25‐hydroxycholesterol (50‐100 μg/ml) result in a 5‐10‐fold increase in cell surface binding sites of high density lipoprotein (HDL). This increase in HDL‐ binding sites was dependent on time and temperature. After a 48‐h exposure to the oxygenated sterol, a maximal increase in HDL. Binding could be observed, and newly induced binding sites disappeared rapidly once 25‐hydroxycholesterol was removed from the medium. No increase in HDL‐binding sites was observed when cells were maintained at 4 °C. In contrast, cultures maintained at 37 °C did show an increase in HDL‐binding sites when exposed to 25‐judrpxycholesterol. Since simulataneous exposure of the cells to 25‐hydroxycholesterol and cyclotheximide resulted in an inhibition of HDL binding to the cells, it is suggested the denovo synthesis of HDL‐binding sites is induced by 25‐hydroxycholesterol. When the abilities of HDL2 and HDL3 to bind to newly synthesized HDL‐binding sites were compared, HDL3 was found to bind more efficiently than HDL2 It is therefore unlikely that apoprotein E plays a major role in the binding of HDL to newly synthesized HDL bind sites were analyzed, they were found to have a high affinity for HDL, since half‐maximal binding was reached at a concentration as low as 5 μg HDL protein/ml, and were saturable. Such HDL‐binding sites had a relaxed specificity, since they were capable of binding low density liprotein (LDL). However, when LDL bound to newly synthesized HDL binding sites, it was no longer internalized, as reflected by a 90% reduction of LDL degradation, and instead of being cytotoxic it became mitogenic.
The cdc 22 Mutation in Schizosaccharomyces pombe is a Temperature‐Sensitive Defect in Nucleoside Diphosphokinase
Authors: DICKINSON J Richard
Published: March 2005

A number of temperature ‐sensitive cdc mutants of schizosaccharomycers pombe that are affected in DNA replication, were screened for the absence of deoxynucleoside triphosphate(s) when blocked at their restrictive temperature. The preliminary screening simply involved analysis of perchloric acid‐soluble cell extracts by two dimensional thin‐layer chromatography on poly(ethyleneimine)‐impregnated cellulose.One mutant strain,cdc 22‐M45, was found which apparently lacked dTTP Pulse‐labelling of intracellular nucleotides revealed that not only did dTTP become depleted, but that dTDP accumulated when this mutant was blocked by a temperature shift‐up, indicating a defective nucleoside diphosphokinase. Nucleoside diphosphokinase from cdc 22‐M45 was less active than that from wild‐type strain 972 when assayed at high temperatures. The nucleoside diphospho kinase of the mutant also has an altered km for dTDP at both permissive(25 C), and at the restrivtive (36.8 C) temperatures. At the restrivtive temperature the Km for dTDP of the mutant enzyme is more than 11‐times greater than that of the wild type. Charaacterisation of the biochemical basis of the defect in this cdc mutant has shown than in S. pombe, despite its having an apparently complex system of genetic control over progression through S‐ phase, one factor at least is merely availability of a nucleoside triphosphate precursor to DNA synthesis.
Ribosomal Phosphoproteins in Acanthamoeba castellanii
Authors: JANTZEN Helga
Published: March 2005

Ribosomes and subunits from Acanthamoeba castellanii were purified. The pattern of ribosomal proteins was analysed by two‐dimensional get electrophoresis. The small subunit of cytoplasmic ribosomes contains 25 and the large 40 proteins. After incubation of exponentially growing cells with ortho[33P]phosphate for 1 j, one basic protein of the small subunit was radiolabelled. This phosphorylated protein has a molecular weigh of about 45000 as estimated by dodecylsulfate/polyacrylamide get electrophoresis. The level of phosphorylated ribosomal protein as estimated by staining of proteins separated by two‐dimensional gel electrophoresis changes in response to growth conditions. The maximum level occurs after adding fresh nutrient medium to cells which have been allowed to grow to high density, whereas ribosomes of exponentially growing cells contain low amounts. Transfer of exponentially growing cells into a non‐nutrient medium induces the formation fo cysts containing ribosimes almost devoid of phosphorylated protein.
Comparison of the Mannan Structure from the Cell‐Wall Mutant Candida sp. M‐7002 and Its Wild Type
Authors: HAMADA Takashi , NAKAJIMA Tasuka , IZAKI Kazuo , MATSUDA Kazuo
Published: March 2005

The cell‐wall mutant of a hydrocarbon‐assimilating yeast, Candida sp. M‐7002 and its wild type have shown a significant difference in mannose content. Each mannan was isolated from the mutant and the wild‐type cells by fractionation with Cetavlon and copper reagent. Both mannans contain d‐mannose, d‐glucose and phosphate. The mutant mannan has a relatively high content of protein (18% in weight bases) whereas the wild‐type mannan has a low protein content (5.1%) with a high amount of carbohydrate (<90%)

Structural analyses by enzymatic and chemical methods showed that both mannans had an mannosidic(α1‐6)‐linked back bone substituted at O‐2 by side chains fo varying length. The side chains of the mutant mannan were shown to consist of single mannose units and disaccharide units whose linkages were predomonstrated that the mannans also contain mannosyl oligosaccharides linked to protein through O‐glycosidic linkage.

The chemical properties of the mannan of the Candida mutant indicates that the mutation might occur not only in the side chain structure but also in the (α1‐6)‐linked mannan back bone.
Comparison of the Mannan Structure from the Cell‐Wall Mutant Candida sp. M‐7002 and Its Wild Type
Authors: HAMADA Takashi , NAKAJIMA Tasuku , MATSUDA Kazuo
Published: March 2005

The structure of the proteomannans from the cell‐wall mutant Candida sp. And its parent strai, was further studied by immunochemical methods. Alkaline titration showed that the phosphate is present in the form of diester and mild acid treatment released mannose from both of the mannans. In the wild‐type mannan, the phosphorylated side‐chain oligosaccharides gave 85% inhibition and a mannopentaose side chain having α1‐3 linkage in the nonreducing terminal also gave 40% inhibition in the homologous precipitin reaction. This indicates that the immunodominant side chain of the Candida sp. Wild type mannan is the phosphorylated mannopentaose which has an(α1‐3)‐linked nonreducing terminal.In the mutant system, a significant reactivity in the cross reaction between the mutant antiserum and Kloeckera brevis mannan indicates that the immunodeterminant of the mutant mannan may be a structural analogue of the side‐chain oligosaccharide from K. brevis which has phosphorylatred(α1‐2)‐linked mannotriose. However, no definite results were obtained in the inhibition studies. On the basis of the results from chemical, enzymatical and immunological experiments, the side‐chain structure and macromolecular models od Candida sp. Wild‐type and mutant mannans are proposed.
tRNA Thiolated Pyrimidines as Targets for Near‐Ultraviolet‐Induced Synthesis of Guanosine Tetraphosphate in Escherichia coli
Authors: THOMAS Gilles , THIAM Khoudia , FAVRE Alain
Published: March 2005

Illumination with near‐ultraviolet light triggers synthesis of ppGpp(guanosine 3;‐diphosphate 5′‐disphosphate) not only in growingEscherichia coli cells containing the putative chromophore 4‐thiouridine in their tRNAs[Ramabhadran, T.V. and Jagger, J.(1976) Proc. Natl Acad.Sci USA, 73, 59‐69], but also in nuv− cells which lack 4‐ thiouridine.

The burst of ppGpp in nuv− cells is, however, induced exclusively by light of wavelengths shorter than 350 nm. Its maximum level is half that obtained in the parental strain. This ppGpp synthesis is also under the control of the relA gene, indicating that it is due to the accumulation of uncharged tRNAs.

A candidate likely to trigger this effect is a 5‐methylaminomethyl‐2‐thiouracil residues present in the fiest position of the anticodon loop of tRNA Glu, tRNALys and one tRNAgln isoacceptor.in conditions in vitro, this base is highly photoreactive at wavelengths shorter than 350nm. Furthermore, near‐ ultraviolet‐photomodified tRNAGlu and tRNALys become poor substrates of their acylation enzyme.
The Specificity of the Sodium Channel for Monovalent Cations.
Authors: FRELIN Christian , VIGNE Paul , LAZDUNSKI Michel
Published: March 2005

The specificity of bthe Na+ channel for monovalent cations has been analyzed in three different ways using neutrotoxins and excitable cells in culture.

Influx experiments using radioactive monovalent cations have shown that all neurotoxins or combination of neurotoxins which change the gating system of the Na+ channel (veratridine, batrachotoxin, dihydrograyanotoxin, scorpion and sea‐anemone toxins) induce a considerable alteration of the permeability of the channel for monovalent cations. Theer is a much decreased ionic selectivity of the channel. Toxin‐induced changes in ionic selectivity are the same or very similar in neuroblastoma cells and in skeletal myotubes. Thefact that toxins which change the kinetics of the gating system also change channel selectivity indicates that the selectivity filter and the gating system may be coupled entities.

Efflux experiments using neuroblastoma cells preloaded with 22N+, 42K+ or 86Rb+ indicate that the toxin stimulated release of these monovalent cations through the Na+ channel is not seen in the presence of cholinium in the external medium. It is seen only if the external medium contains permeant monovalent cations. The ‘chemical’ activation of the Na+ channel by neurotoxins necessitates the saturation at the external face of the channel of sites specific for permeant monovalent cations. Dose‐response curves for the effect of increasing external concentrations of Na+, K+, Rb+ or guanidinium on 22Na+ efflux indicate positive cooperativity in the stimulating effect of these cations. The best apparent affinity is observed for guanidinium, then for Na+, K+ and Rb+.

Competition experiments between monovalent cations and a radiolabelled tritiated derivative of tetrodotoxin for binding to the tetrodotoxin receptor of the Na+ channel in neuroblastoma cells indicatae that monovalent cations bind cooperatively to external sites for Na+. The Hill coefficients and the dissociation constants for Na+ and guanidinium binding to the Na+ channel measured with this approach compare well to values found from 22Na+efflux experiments.
Cloning of Four DNA Fragments Complementary to Human Thyroglobulin Messenger RNA
Authors: BERGÉ‐LEFRANC Jean‐Louis , CARTOUZOU Guy , MALTHIÉRY Yves , PERRIN Fabienne , JARRY Bruno , LISSITZKY Serge
Published: March 2005

Human thyroglobulin mRNA was isolated from Graves' goiters by size selection of total poly(A)‐rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis.cDNA was synthesized from the 33‐S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double‐stranded using reverse transcriptse and blunt‐ended with nuclease S1.After tailing with dCTP and terminal transferase, the double‐stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP.The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33‐S mRNA, visualization of cDNA mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400‐1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs.Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
Endoplasmic Reticulum Nuclease
Authors: KOUIDOU Sofia , TRIANTOS Althanasios , KAVOUKOPOULOS Evangelos , TRAKATELLIS Antonios
Published: March 2005

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity was isolated from rat liver endoplasmic reticulum.The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 × 104,9 × 104,1.55 × 105 and Sepharcyl S‐200 fraction at V0) Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphare electrophoresis (Mr5.4 × 104). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights.

Deoxyribonucleoside and ribonucleoside triphophates can bind to the endoplasmic reticulum nuclease, Binding is enhanced in the presence of divalent cations particularly Mg2.The enzyme exhibits mainly Rnase activity but can also degrade denatured DNA and DNA RNA hybrids which contain breaks in one of the two starands. Poly (A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.
Domain Structure of Isocitrate Dehydrogenase from Azotobacter vinelandii
Authors: SLOBBE Wilma , VOORDOUW Gerrit
Published: March 2005

Isocitrate dehydrogenase from Azotobacter Vinelandii, which consists of a single polypeptide chain of Mr 80,000, is completely unfolded in 3 M guanidine hydrochloride at pH 7.5 and 22 C as judge by hydrodynamic criteria and a study of tryptophan fluorescence.

Transfer of denatured isocitrate dehydrogease to native solution conditions (less than 0.1 M guanidine hydrochloride) leads to rapid reactivation (t1/2 ∼ 1 min at 22 C,pH 7.5). Practically complete reactivation (85‐95%) can be achieved under properly chosen conditions.

At high protein concentration(c > 0.5 ‐ 1.0 mg/ml) intermolecular aggregation competes with proper folding and reactivation. Renatured isocitrate dehydrogenase is indistinguishable from the original native enzyme provided that aggregated material is removed by centrifugation and gel filtration.

The denaturation by guanidine hydrochloride proceeds in two separable steps: N⇄D1⇄D2.The midpoint of the N⇄ D1 transition is at 0.3 M, that of the D1⇄ D2 transition at 1.2‐1.5 M guanidine hydrochloride. The sedimentation coefficient (s20,w) drops from 4.5 S for the native conformation N to 4.5 S for D1 and 2.3 S for D2 The relative values for the tryptophan fluorescence of D1 and D2 are 50% and 25% of that of N, respectively (γex = 295nm, γem = 330nm).

Protease digestion studies using thermolysin or trypsin or trypsin in 0.4 M guanidine hydrochloride suggest that isocitrate dehydrogenase from A vinelandii is composed of three domains which, according to their molecular masses, are designated as the 45‐kDa and the 19‐kDa, the 16‐kDa domains. The 45‐kDa and 16‐kDa domains are resistant to protease digestion in 0.4 M guanidine hydrochloride, while the 19‐kDa domain is rapidly digested under these conditions.

The(un)folding intermediate D1 thus represents a conformation comprising folded 45‐kDa and 16‐kDa domains and a largely unfolded 19‐kDa domain. In D2 all three domains have unfolded. The observation that intermolecular aggregation is favored by low concentrations of guanidine hydrochloride (0.3‐0.5 M) indicates that this process may depend on intermolecular interactions between the 45‐kDa and 16‐kDa domains.
Intraparticulate Localization of Some Peroxisomal Enzymes Related to Fatty Acid β‐Oxidation
Authors: HAYASHI Hidenori , HINO Satoru , YAMASAKI Fumie
Published: March 2005

Male Wistar rats were given a diet containing 0.05 % (w/w) LK‐903 (α‐methyl‐p‐myristyroxycinnamic acid 1‐ monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl‐CoA β‐oxidizing system were determined. The activities of fatty acyl‐CoA oxidase, crotonase, β‐hydroxybutyryl‐CoA dehydrogenase and thiolase were all increased about three times by administration of LK‐903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X‐100, by sonication, and by sucrose‐density‐gradient centrifugation after Triton X‐100 treatment. The results suggesl that thiolase is localized in the matrix of peroxisomes, that crotonase and β‐hydroxybutyryl‐CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl‐CoA oxidase is associated with the core, though its association is weak.
Ganglioside Biosynthesis in Rat Liver
Authors: SENN Hans‐Jürgen , COOPER Cecil , WARNKE Peter C. , WAGNER Marita , DECKER Karl
Published: March 2005

UDP‐N‐acetylgalactosamine–GM 3acetylgalactosaminyltransferase(GM 2‐synthase) was studied in a Golgi‐rich fraction from rat liver. Activity in a cell‐free system required the presence of detergents; octyl glucoside was found to be the most effective in stimulating the enzyme. Optimal activity of GM 2‐synthase was obtained at pH 7.2, in the presence of 0.8% octyl glucoside, 10 mM Mn2+and 5 mM CDP‐choline. The latter was used to counteract the rapid sugar nucleotide hydrolysis caused by a nucleotide pyrophosphatase activity in the Golgi fraction. The apparent Kmvalue for UDP‐N‐acetylgalactosamine was 0.18 mM and Co2+and Fe2+exceeded Mn2+in activating GM 2‐synthase. Under optimal assay conditions and in the presence of added GM 3and 5 mM CDP‐choline, the specific activity of the enriched Golgi fraction was measured to be 25–30 nmol × mg protein−1× h−1; with endogenous GM 3as the sole glycolipid acceptor, V was calculated to be 9 nmol × mg protein−1× h−1.
Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarum polycephalum
Authors: BELANGER Gilles , GODIN Claude , LEMIEUX Gérald
Published: March 2005

The rate of phosphate exchange in individual ribosomal proteins of Physarum polycephalum was determined in vivo. It was observed that the phosphoryl groups of S3, the major phosphoprotein, had a turnover rate of 1.5% per minute. The phosphorylation of ribosomal proteins is regulated by at least two different mechanisms. The rapid turnover of phosphyl groups of the major phosphoprotein is in agreement with the general observation that the phosphate content of this protein is modulated by the physiological state of the cells and possibly involved in the regulation of ribosome activity. The absence of phosphate exchange in accidic proteins suggests that these groups could play a structural role in the ribosome functions.
Quasi‐Lipoxygenase Activity of Haemoglobin
Authors: KÜHN Hartmut , GÖTZE Regina , SCHEWE Tankred , RAPOPORT Samuel M.
Published: March 2005

Haemoglobin catalyses at low concentrations (0.01–1 μM) a quasi‐lipoxygenase reaction with remarkably high substrate specificity. Formation of lipohydroperoxides was demonstrated only with free 9‐cis,12‐cis‐octadecadienoic acid (Linoleic acid), its glycerol monoester, or 9‐cis,12‐cis‐octadecadienol (Linoleoyl alcohol). All‐cis,‐9,12, 15‐octadecatrienoic acid (α‐linolenic acid), all‐cis,‐5,8,11,14‐eicosatetraenoic acid (arachidonic acid) and all‐cis,‐9,12, 15‐ eicosatetraenoic acid (dihomo‐ν‐linolenic acid), phospholipids and biological membranes were not attacked. Saturated and unsaturated free fatty acids other than linoleic acid were strong inhibitors of the haemoglobin‐catalyzed oxygenation of linoleate. The reaction was also inhibited by cyanide, carbon monoxide, phenolic antioxidants and the lipoxygenase inhibitor salicylhydroxamic acid, as well as by some anti‐inflammatory drugs (prednisolone, dexamethasone, acetylsalicylic acid); indomethacin did not inhibit. Protein inhibitors of liproxygenase reactions were found in lung homogenates of various species. The molecular activity of the haemoglobin‐catalyzed oxygenation of linoleate was comparable with those of true lipoxygenases and was strongly suppressed by denaturation (heating, urea, pronase treatment, acid splitting). The apparentS0.5, the activation energy and the pH‐optimum did not differ from those of most of the true lipoxygenases.

The reaction product hydroperoxylinoleic acid but not hydrogen peroxide was an activator of the haemoglobin‐catalyzed oxygenation of linoleate. Hydroxylinoleic acid was a competitive inhibitor of the activation. At haemoglobin concentrations higher than 1 μM the quasi‐lipoxygenase activity was completely suppressed presumably owing to the formation of inhibitory concentrations of hydroxylinoleate via the lipohydroperoxidase [21] activity of haemoglobin.

The quasi‐lipoxygenase reaction of haemoglobin exhibits a suicidal behaviour, caused by destruction of haem groups. The properties of the free haemin‐catalyzed reaction were similar to those of haemoglobin. Among other haemoproteins tested myoglobin and cytochrome P‐450LMshow comparable activities. The mechanism of the haemoglobin‐catalyzed oxygenation of linoleate seems to involve a change of valency of the haem iron.
Storage Proteins in Zea mays (L.): Interrelationship of Albumins, Globulins and Zeins in the Opaque‐2 Mutation
Authors: DIERKS‐VENTLING Christa
Published: March 2005

Mature endosperms from opaque‐2 mutant seeds with barious genetic backgrounds (B 37, W 64A, R 802A and Oh 43) contained twice as much globulin than the corresponding normals, reduced amounts of zein and an increased amount of albumin. The letter is caused by a diminished disappearance of albumins in mutant endosperms during the final phase of development as compared to normal endowperms. Albumins from the opaque‐2 endosperms appeared on gel electrophoresis as a similar but heterogenous polypeptide population comparable to that of the normal endosperms except that in al opaque‐2 forms a 70‐kDa polypeptide was increased. Similarly, one to three specific globulin polypeptides (47 kDa, 52 kDa and 58 kDa) were, depending on the genotype, increased in the mutant lines. The accumulation of albumins, globulins and zeins, studied in developing W 64A opaque‐2 mutant and corresponding normal maize kernels from 10 days after pollination until maturity, demonstrated two‐phasic accumulation patterns for all proteins in the mutant, the first phase extending to about 30 days post pollination and the second one form there on until maturity. The transition time‐point from first to second phase was characterized in mutant endosperm by a sudden reduction in accumulating albumins (also seen in normal endosperm), by an enhanced accumulation of globulins and the cessation of further accumulation of zein. Preferential accumulation of certain globulins has been found in the mutant during the second phase of globulin formation. The increased accumulation of globulins in the opaque‐2 mutant endosperm is considered a response to the arrest in zein synthesis.
A New Principle of Regulation of Enzymic Activity
Authors: TSCHESCHE Harald , MACARTNEY Henry W.
Published: March 2005

Latent collagenase form human polymorphonuclear leukocytes was isolated and shown to be activatable by disulfides, e.g. cystine, oxidized glutathione and insulin. Activation proceeds via a disulfide‐thiol exchange mechanisms by which the active proteinase (Mr 65500–67000) is released from the inactive latent enzyme (Mr 91000–94000) which is a mixed disulfide of an inhibitor (Mr20000–25000) and the collagenase [Macartney and Tschesche (1980) FEBS Lett. 119, 327‐332].

In a system in vitro the activation can be coupled to the glutathione cycle. Reduced glutathione and hydrogen peroxide in the presence of any of the enxymes glutathione peroxidase, NADH peroxidase, lactoperoxidase, horse radish peroxidase or human leukocyte myeloperoxidase activate the latent collagenase. Pre‐incubation of the peroxidase with the inhibitor sodium azide prevents the activation of the latent enzyme via hydrogen peroxide and reduced flutathione. The oxidizing equivalents can also be generated by employing the hydrogen‐peroxide‐generating system of glucose and glucose oxidase. The peroxidase‐catalysed activation by the glucose/glucose oxidase system can be inhibited by including either catalase or glutathione reductase in the activating system. NADH also inhibits the glucose‐coupled activation system, but this effect can be counteracted by including NADH peroxidase.

The result indicate that the activation of the latent collagenase can be regulated by the glutathione cycle. The activation could be coupled in vitro to the glucose metabolism via the system glucose/glucose oxidase. Regulation in vitro might perhaps be coupled to the phagocytosis‐associated respiratory burst known to be linked to the hexose monophosphate shunt activity of the cell.

In an experiment in vitro, with the concentrations of reduced and oxidized glutathione normally found in vivo, the glutathione cycle could be used either to activate or to inactivate the latent collagenase, depending on the addition of either hydrogen peroxide as oxidizing equivalents or NADPH or NADH as reducing equivalents. This provides the first example in which regulation of proteolyticactivity activity could be linked to the glutathione redox potential of the cell.
Bovine Tryptophanyl‐tRNA Synthetase
Authors: KISSELEV Lev L. , FAVOROVA Ol'ga O. , NURBEKOV Malik K. , DMITRIYENKO Stanislava G. , ENGELHARDT Wladimir A.
Published: March 2005

As is found by atomic absorption spectroscopy, the highly purified bovine tryptophanyl‐tRNA synthetase contains up to 0.9mol Zn+2/mol enzyme while some other bivalent metal ions are absent. The enzyme is inactivated either upon treatment with 1,10‐phenanthroline(a zinc‐chelating agent) or upon prolonged dialysis (which eliminates bound Zn+2 ions); addition of zinc reativates the enzyme. Exposed histidine residues(s) and carboxylic group(s) of the enzyme are involved in the Zn+2 binding, as shown using chemical modification. Circular dichroism spectra suggest that elimination of Zn+2 ions affects the tertiary rather than the secondary structure of the tryptophanyl‐tRNA synthetase. The kinetics of inhibition with 1,10‐phenanthroline toward ATP, tryptophan and tRNATrp indicates that removal of zinc prevents the ATP binding to the enzyme.
The Charge Heterogeneity of the Mitochondrial Acetyl‐CoA Acetyltransferase from Rat Liver
Authors: HUTH Walter
Published: March 2005

The charge heterogeneity of the mitochondrial acetyl‐Coa acetyltransferase (EC 2.3.1.9), referred to transferases A and B, seems to be performed in vivo and can be demonstrated in vivo by a spontaneous transformation of transferase A into transferase B. these two enzymes, although remarkably similar in amino acid composition, show structural dissimilarities. This becomes evident from their tryptic maps, which are substantially different.

Isoelectric focusing in the presence of urea confirms the charge heterogeneity of the mitochondrial acetyl‐CoA acetyltransferase by indicating different patterns of protein bands for transferases A and B. transferase B lacks the protein band with an isoelectric point of 7.2 which is normally shown with transferase A. However, this 7.2‐pI protein band can be demonstrated after a [3H]CoASH treatment of transferase B in the presence of urea by protein sustaining and by autoradiography. This change in the isoelectric focusing band pattern after interaction of the subunits with CoASH is interpreted as being the result of an apparently covalent secondary modification of the protein side chains.

The CoASH‐modification may be the only molecular basis of the acetyl‐CoA acetyltransferase change heterogeneity, a view, which is further substantiated by the CoASH‐mediated transformation of transferase B into transferase A. An additional heterogeneity of the enzyme as caused by a limited proteolysis seems unlikely but cannot be definitely excluded.
Binding of Magnesium and Chloride Ions to Human Hemoglobin A
Authors: ACHILLES Wolfgang , CUMME Gerhard A. , WINNEFELD Klaus , FRUNDER Horst
Published: March 2005

Ultrafiltration and direct determination of [Mg2+]by ion exchange were used to study the binding of Mg2+ and Cl− to isoionic human hemoglobin

A value of 58.8+1.71/mol was determined for the volume of hydrated hemoglobin from ultrafiltration of hemoglobin solutions containing 0.15–0.8 M glucose.

In solutions with 5.7 mmol hemoglobin,150mmol KCl and 0.5–3.5 mmol MgCl2/l total water,0.6 mol and 2 mol cl− were bound/mol oxygenated and deoxygenated hemoglobin respectively. A value of about 111/mol was determined for the association constant of Mg2= to hemoglobin monomer.

Free Mg2+ concentrations were measured in hemoglobin solutions containing KCl, MgCl2, ATP and d‐ glycerate‐2.3‐bisphosphate at concentrations close to those of red cells. The experiments yielded 0.65 mmol/l free Mg2+ after oxygenation and 0.82 mmol/l after deoxygenation.The data indicate that only small changes of free Mg2+ levels in red cells are caused by physiological changes of pO2.
NADH Dehydrogenase and NADh Oxidation in Membrane Vesicles from Bacillus subtilis
Authors: BERGSMA Jack , STRIJKER Rein , ALKEMA Jet Y. E. , SEIJEN Hendrik G. , KONINGS Wil N.
Published: March 2005

Crossed immunoelectrophoresis of membrane vesicles of Bacillus subtilis W23, solubilized in 5% Triton X‐100, against anti‐vesicle imunoglobulins resulted in at least 43 immunoprecipitates.With the zymogram staining techique the precipitation lines of the enzymes succinic dehydrogenase, malate dehydrogenase, ATPase, NADPH dehydrogenase and NADH dehydrogenase were identified.

NADH‐dehydrogenase activity was found in three precipitation lines. Preparative polyacrylamide gel electrophoresis of NADH dehydrogenase, solubilized with 0.1% Triton X‐100, showed three to five major protein bands with NADH dehydrogenase activity.The sodium dodecyl sulphate/polyacrylamide gel electrophoresis patterns of these bands all contained a band of the catalytic subunit with an apparent Mr of 64000. Antibodies against this catalytic subunit were prepared. The presence of only one NADH dehydrogenase in the cytoplasmic membrane was indicated by the facts that solubilized (NADH dehydrogenase) immunoglobulins and that membranes solubilized with 5% Triton X‐100 showed the same three precipitation lines with NADH dehydrogenase activity when either anti‐(NADH dehydrogenase) or anti‐(membrane vesicle) immunoglobulins were used.

This NADH dehydrogenase is located at the inner surface of the cytoplasmic membrane(like succinic dehydrogenase and ATPase) as was shown with immunoabsorption experiments.

Membrane vesicles oxidize NADH, presented at the outer surface, at a high rate. The mechanism of NADh oxidation was studied.NADH is oxidized via the respiratory chain. Uptake of NADH by the membrane vesicles could not be demonstrated and ermoval of 74% of NADH dehydrogenase resulted only in a 24% decrease of NADH oxidase activity. Membrane vesicles from the menaquinone‐deficient strain B subtilis aroD do not oxidize externally added NADH but BADH oxidation can be restord by addition of the menaquinone analogue, menadione. These resuts suggest that externally added NADH donates electrons directly to menaquinone‐7 at the outer surface of the vesicle membrane and that NADH dehydrogenase is not involved in this oxidation process.
Tightly Bound Non‐histone Proteins in Nucleosomes from Pig‐Liver Chromatin
Authors: CAIAFA Paola , SCARPATI‐CIOFFARI M. Rosaria , ALTIERI Fabio , ALLEGRA Paola , LONIGRO Renata , TURANO Carlo
Published: March 2005

Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non‐histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71 000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two differ‐ ent classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are com‐ patible or incompatible, respectively, with the simultaneous binding of the histone octamer.
The Isolation, Mapping and Transcription in vitro of a β0‐Thalassaemia Globin Gene
Authors: JACKSON Ian J. , FREUND Rhayza Maingon , WASYLYK Bohdan , MALCOLM Alan D. B. , WILLIAMSON Robert
Published: March 2005

The red blood cell precursors of a patient with homozygous β°‐thalassaemia have previously been shown to contain nuclear, but not cytoplasmic, β‐globin‐specific transcripts. We describe the isolation of a β‐globin gene from this patient as a recombinant bacteriophage chromosome. Restriction‐enzyme cleavage‐site mapping expcriments demonstrate no detectable deletions, insertions or major rearrangements in this thalassaemia gene. Two different techniques show that the gene isolated is transcribed as efficiently in vitro as the normal β‐globin gene.
Biosynthesis of Pyruvate Kinase Isozymes in Rat Liver
Authors: WU Stephen Wing Nin , WONG Susanna Siu Chun , YEUNG Desmond Chak Yew
Published: March 2005

The L and M1 isozymes ofpyruvate kinase were purified to homogeneity from rat liver and muscle respectively and their specific antibodies were employed to quantify the isozyme concentration in rat liver during development.

Total enzyme activity decreases towards birth and reaches a minimum on the 3rd postnatal day, but the activity increases dramatically after weaning.

Immunoprecipitation revealed that the M2 type predominates in the prenatal period but decreases sharply just before birth.

The L isozyme contribution is augmented upon weaning and is sustained until the rat is adult and a L/M ratio of 9:1 is maintained.

By means of incorporation studies with [3H]leucine followed by immunoprecipitation, the increase in L‐type activity when approaching term and after weaning is explained by a twofold increase in its rate of synthesis coupled with a concomitant reduction of the M2‐type synthesis.