bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Studies of Adenovirus Type‐2 Hexon Protein
Authors: JÖRNVALL Hans , PETTERSSON Ulf , PHILIPSON Lennart 
Published: March 2005

The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2‐14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical.Structural analysis of [14C]carboxymethylated hexon and of hexon which was labeled in vivo with [35S]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100 000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments.Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained.
Carbon Monoxide Oxidation by Growing Cultures of Clostridium pasteurianum
Authors: FUCHS Georg , SCHNITKER Ursula , THAUER Rudolf K. 
Published: March 2005

Growing cultures of Clostridium pasteurianum were found to oxidize carbon monoxide to CO2 at a maximal rate of approximately 15 nmol × min−1× mg protein−1. The dependence of the rate on the carbon monoxide concentration followed simple Michaelis‐Menten kinetics. Half‐maximal velocity was obtained with 3 μM carbon monoxide in the growth medium. The values of V and[S]0.5V determined in vivo are similar to those reported for carbon monoxide oxidation with flavin nucleotides in cell‐free extracts of C. pasteurianum (V= 14 nmol × min−1× mg protein−1; [S]0.5V= 5 μM), indicating that in vivo and in vitro carbon monoxide is oxidized by the same system.Carbon monoxide was even removed from the medium by growing cultures of C. pasteurianum at concentrations as low as 1 nM equal to a concentration in the gas phase of approximately 1 part per million. Carbon monoxide was oxidized at a rate of 8 μmol × 1−1× h−1 under these conditions. The biological significance of this finding is discussed.
Antagonism of Insulin and Lipolytic Hormones in the Control of Adenylate‐Cyclase Activity in Fat Cells
Authors: RENNER Rolf , KEMMLER Wolfgang , HEPP K. Dieter 
Published: March 2005

The control of adenylate cyclase activity by lipolytic hormones was studied in fat cell “ghosts” in the presence and absence of physiological concentrations of insulin. Insulin was found to inhibit the effect of submaximal concentrations of norepinephrine, adrenocorticotrophin, and glucagon. Addition of the hormone to the assay system leads to an immediate reduction of the linear rate of formation of adenosine 3′:5′‐monophosphate (3′:5′‐AMP). For norepinephrine and adrenocorticotrophin a dose‐response relationship was established which was shifted by insulin in a “pseudocompetitive” fashion. Increasing the concentration of insulin beyond the physiological range resulted in a reversal of its inhibitory effect in the presence of lipolytic hormones. Insulin alone at concentrations between 5 and 5000 μU/ml had no effect, whereas a small stimulatory effect of high insulin concentrations (5–500 mU/ml) could be found in the presence of submaximal concentrations of norepinephrine. No effect of insulin was obtained against several concentrations of NaF. The effect of insulin upon the norepinephrine‐stimulated adenylate cyclase disappeared after trypsin treatment of intact cells prior to preparation of ghosts. Further incubation of intact fat cells after trypsin treatment lead to a recovery of the effect of insulin. Analogous to insulin, nonsuppressible insulin‐like material which is believed to be related to “somatomedin”, was found to inhibit adenylate cyclase activity in ghosts, but this effect was not abolished by trypsin treatment. Ghosts were found to contain 3′:5′‐AMP phosphodiesterase activity which was not influenced by direct addition of insulin as measured with low substrate concentrations or under the conditions of the adenylate cyclase reaction.It is suggested that insulin at low concentrations interferes with the activation of the adenylate cyclase system by lipolytic hormones, at a step between hormone receptor and catalytic unit. In view of similar characteristics of the antilipolytic effect of insulin in intact lipocytes and the observed modulation of the action of lipolytic hormones in ghosts, a fundamental physiological role of this effect upon the adenylate cyclase system in the mechanism of action of insulin is postulated.
Regulatory Behavior of Monomeric Enzymes
Authors: MEUNIER Jean‐Claude , BUC Jean , NAVARRO André , RECARD Jacques 
Published: March 2005

The wheat‐germ hexokinase L1 is made up of one polypeptide chain only and exhibits one active site for glucose per enzyme molecule as shown by equilibrium dialysis experiments. However, MgATP2− cannot be bound to a significant extent on the free enzyme in the absence of the hexose.Plots of the reciprocal of the reaction rate versus the reciprocal of glucose concentration exhibit a significant downward curvature, but they are linear versus the reciprocal of MgATP2− concentration. The curvature of the plots with regard to glucose is modified neither by the concentration of MgATP2− nor by that of the product MgADP−. The intercepts and the slopes of the straight lines obtained versus the reciprocal of MgATP2− concentrations give, when replotted against the reciprocal of glucose concentration, a curve concave downwards and a straight line, respectively. The downward curvature of the reciprocal plots, obtained with regard to glucose, is strongly increased by glucose 6‐phosphate.The whole set of kinetic results along with equilibrium dialysis data strongly suggest that the non‐Michaelian behavior is due to a mnemonical transition of the wheat germ hexokinase L1. The free enzyme very probably exists in two conformation states. The collision of any of these enzymes forms with glucose induces the same new conformation. After the binding of MgATP2−, which is thus the second substrate, and the release of MgADP−, the enzyme appears in one of the initial conformations which is stabilized by glucose 6‐phosphate. That ligand can thus be bound on one only of the two conformation states of the free enzyme.The view that wheat germ hexokinase L1 is a mnemonical enzyme gains further support from the study of the effect of denaturing agents on the reaction kinetics. Low concentrations of urea or dodecylsulfate “desensitize” the enzyme and completely suppress its non‐Michaelian behavior, which clearly appears as a consequence of a conformational transition.
Involvement of DNA Polymerases I and III in the Replication of Bacteriophage M‐13
Authors: STAUDENBAUER Walter L. 
Published: March 2005

M‐13 replication was investigated in Escherichia coli mutants deficient in DNA polymerase I (polA) and DNA polymerase III (dnaEts). The following results were obtained:

M‐13 infection is abortive in both polA− and dnaEts mutants at 42°C.
The conversion of the parental single‐stranded DNA to double‐stranded replicative forms (RF) is only slightly affected by the dnaE mutation. However, no parental double strands are formed in the polA− dnaEts double mutant at the restrictive temperature, indicating a possible involvement of DNA polymerase I in this process.
The replication of the replicative‐form molecules as well as the synthesis of progeny single strands are inhibited by thermal inactivation of the dnaE function implying a requirement of DNA polymerase III in both types of DNA synthesis.
The presence of the polA mutation leads to an accumulation of open‐circular replicative forms. DNA polymerase I might, therefore, also be involved in the filling of gaps in the newly synthesized DNA molecules.
On the Mechanism of Translocation of Pyruvate and Other Monocarboxylic Acids in Rat‐Liver Mitochondria
Authors: PAPA Sergio , PARADIES Giuseppe 
Published: March 2005

Pyruvate equilibrates, like other carboxylic acids, across the membrane of rat‐liver mitochondria according to the transmembrane pH difference.
Comparison of the rate of pyruvate, acetoacetate and acetate efflux from mitochondria as well as a study of the effect of mersalyl and N‐ethylmaleimide on this process, show that the diffusion of pyruvic and acetoacetic acid across the mitochondrial membrane is carrier‐mediated. The process might consist of pyruvate (acetoacetate) hydroxyl ion exchange‐diffusion or of pyruvic acid (acetoacetic acid) uniport. On the contrary acetic acid appears to diffuse freely across the membrane.
The initial rate of the net pyruvate uptake by rat liver mitochondria follows saturation kinetics. The increase of the external pH of the medium as well as the addition of acetoacetate inhibit competitively pyruvate uptake. Pyruvate uptake is inhibited by mersalyl and N‐ethylmaleimide.
Mitochondria1 [14C]pyruvate exchanges with unlabelled pyruvate and acetoacetate but not with other monocarboxylates, Pi and citric‐acid cycle intermediates.
The pyruvate‐pyruvate and pyruvate‐acetoacetate exchanges follow saturation kinetics and exhibit the same V but different Km values. Both the exchanges are inhibited by mersalyl, but not by N‐ethylmaleimide.
It is concluded that pyruvate translocation across the mitochondrial membrane is mediated by a specific transport system.
The Inhibition of Proteinases by Human α1‐Antitrypsin
Authors: HERCZ Albert 
Published: March 2005

The inhibition of human plasmin by α1‐antitrypsin was time dependent and the rate of inhibition rapidly decreased with decreasing temperature.The effect of substrates on the inhibitions of trypsin and chymotrypsin was investigated. Theoretically the final degree of inhibition should be the same, whether the substrate is added to the proteinase‐inhibitor system before or after the formation of the complex. The inhibition of chymotrypsin by α1‐antitrypsin and of trypsin by commercial soybean trypsin inhibitor was in line with the prediction, with either casein or synthetic esters as substrates. However, in the trypsin‐α1‐antitrypsin system the degree of inhibition depended on the order in which the reactants were mixed. With both casein and p‐tosyl‐l‐arginine methyl ester as substrates, inhibition was high when trypsin was preincubated with the inhibitor and the substrate was added later, and low or absent when the proteinase was added to the mixture of inhibitor and substrate.In the inhibition mixtures of plasmin, trypsin and chymotrypsin with α1‐antitrypsin, distinct changes were detected by electrophoresis in polyacrylamide gel. The unreacted inhibitor diminished or disappeared and reaction products were formed at rates corresponding to rates of inhibition.
Cyclic AMP and Catabolite Repression in Yeasts
Authors: SCHLANDERER Gustav , DELLWEG Hanswerner 
Published: March 2005

In Schizosaccharomyces pombe an inverse proportionality between glucose concentration and intracellular adenosine 3′:5′‐monophosphate (CAMP) was established. The cAMP levels were in the range of 12 to 785 pmol per g yeast cells dry weight. In continuous cultures a steady‐state concentration of as low as 0.05% glucose is sufficient to cause a decrease in intracellular cAMP content. Lowest levels of CAMP were found in the presence of more than 5% glucose, while highest levels were synthesized by cells grown on glycerol or maltose.In all strains tested, CAMP is transported into the fermentation broth, both if grown on glucose or cultured on maltose as the sole carbon source.The synthesis of α‐glucosidase (EC 3.2.1.20) is induced by maltose. Glucose represses the synthesis of invertase (EC 3.2.1.26), NADH‐ferricyanide reductase (EC 1.6.99.3) and both the constitutive and the inducible α‐glucosidase. The possibility of a coordinate synthesis of α‐glucosidases and invertase is indicated by the experimental data but cannot definitely be proved.Moreover, there exists a relation between the intracellular concentration of cAMP and the activities of invertase, α‐glucosidases and a NADH‐ferricyanide reductase. Under comparable growth conditions a mutant strain partially resistant to catabolite repression synthesizes more cAMP and produces increased activities of the catabolite‐sensitive enzymes tested than the wild‐type strain. Strains classified as catabolite‐sensitive show reduced values of intracellular cAMP and enzyme activities. In contrast to Saccharomyces cerevisiae in Schizosaccharomyces pombe there exists no relation between the intracellular level of cAMP and the activity of alcohol dehydrogenase (EC 1.1.1.1).We suppose, that in Schizosaccharomyces pombe cAMP is concerned with the regulation of catabolite‐sensitive enzyme synthesis.
Biochemistry and Pharmacology of the Crotoxin Complex
Authors: BREITHAUPT Henning , RÜBSAMEN Klaus , HABERMANN Ernst 
Published: March 2005

Crotapotin and the basic crotalus phospholipase A, which are the main components of the crotoxin complex, have been purified to homogeneity with respect to their behaviour on polyacrylamide gel electrophoresis, cellogel electrophoresis, immunoelectrophoresis, isoelectric focusing, and sedimentation equilibrium analysis.The isoelectric points were found to be 3.4 for crotapotin and 9.7 for the crotalus phospholipase A.The molecular weight of phospholipase A has been determined by gel filtration in 6 M guanidine · HCl (14500), by ultracentrifugation (15800), and by dodecylsulfate‐gel electrophoresis (15800). Phospholipase A consists of a single peptide chain intramolecularly crosslinked by eight disulfide bridges.The molecular weight of crotapotin was 8900 as shown by gel filtration in 6 M guanidine · HCl. Sedimentation equilibrium studies in 4 M guanidine · HCl gave a value of 6700. In solutions of lower ionic strength, the Mr, was 12500, indicating that crotapotin can exist as a dimer. In contrast the Mr of reduced and carboxamidomethylated crotapotin was much lower (4000–4500), in gel filtration experiments.Crotapotin consists of three peptide chains, held together by disulfide bridges. Chain A contains 40 amino acid residues (Mr 4274), chain B 34 residues (Mr 3658) and chain C 14 residues (Mr 1558). A free NH2‐terminal residue (serine) could be detected only for chain A.
Interaction between Arginase and l‐Ornithine Carbamoyltransferase in Saccharomyces cerevisiae
Authors: PENNINCKX Michel , SIMON Jean‐Paul , WIAME Jean‐Marie 
Published: March 2005

l‐Ornithine carbamoyltransferase and arginase from Saccharomyces cerevisiae have been extensively purified and their quaternary structures have been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Subunit cross‐linking with glutaraldehyde shows that both enzymes have trimeric structures. Ornithine carbamoyltransferase is built up by the association of three subunits of molecular weight 37500; arginase is made of three subunits of 39000. The latter observation has lead us to reinvestigate the quaternary structure of rat liver arginase for which, in spite of an earlier report to the contrary [H. Hirsch‐Kolb and D. Greenberg, J. Biol. Chem. 243, (1968) 6123–6129], evidence of a trimeric structure is also obtained.The data suggest that the 1‐to‐1 regulatory complex of ornithine carbamoyltransferase and arginase of S. cerevisiae, leading to ornithine carbamoyltransferase inhibition, is an hexamer.
Molecular Forms of Rat‐Liver Arginase. Isolation and Characterization
Authors: TARRAB Rebeca , RODRÍGUEZ Jesús , HUITRÓN Carlos , PALACIOS Rafael , SOBERÓN Guillermo 
Published: March 2005

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms.After 2500–5000‐fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl‐cellulose columns and were purified 2500–5000‐fold, 800–1000‐fold and 600–1000‐fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose‐lysine as a competitive inhibitor of arginase, shows only one main form, however.Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea) the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea, between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin‐layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants.
The Synthesis of Adenine‐Substituted Derivatives of NADP+ and Their Potential as Active Coenzymes and Affinity Adsorbents
Authors: LOWE Christopher R. , MOSBACH Klaus 
Published: March 2005

Alkylation of NADP+ with iodoacetic acid at pH 6.5 yielded N1‐carboxymethyl‐NADP+ which, following enzymic reduction, alkaline rearrangement and enzymic reoxidation, gave N6‐carboxy‐methyl‐NADP+. Condensation of these analogues with 1,6‐diaminohexane in the presence of a water‐soluble carbodiimide generated the N1 and N6N‐(6‐aminohexyl)‐acetamide derivatives of NADP+ respectively. The coenzymic activities of the N1 and N6 adenine‐substituted carboxymethyl and N‐(6‐aminohexyl)‐acetamido derivatives of NADP+ were tested with several NADP+‐dependent dehydrogenases. The carboxymethyl derivatives were more active (up to 145%) relative to un‐substituted NADP+ than the corresponding N‐(6‐aminohexyl)‐acetamido compounds (up to 10%).NADP+ ‐N6‐[N‐(6‐aminohexyl)‐acetamide] was coupled to a soluble dextran and to Sepharose 4B by the cyanogen bromide technique. The dextran‐bound derivative contained 85 μmol NADP+ analogue/g dry dextran and was reduced at approximately 35% the rate of native NADP+ with glucose‐6‐phosphate dehydrogenase. The dextran‐bound NADP+ cycled at a rate of about 30% relative to free NADP+ in a system comprising glucose‐6‐phosphate dehydrogenase and l‐glutamate dehydrogenase. Sepharose‐bound NADP+ proved to be an effective biospecific adsorbent for affinity chromatography and was competent for the resolution of a mixture of bovine albumin, lactate dehydrogenase and glucose‐6‐phosphate dehydrogenase. The latter was eluted almost quantitatively with a pulse of 0.5 mM NADP+.
A New Subgroup of Human L‐Chains of the λ‐Type
Authors: EULITZ Manfred 
Published: March 2005

Bence‐Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion‐exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion‐exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic peptides were isolated and characterized to obtain overlapping peptides for the whole V‐region and for some tryptic peptides of the C‐region.Protein DEL is related to subgroups III and IV of the λ‐chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups III and IV but in contrast to subgroups I and II of the λ‐chains. However, marked differences exist between protein DEL and the proteins of subgroups III and IV.a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL.b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the λ‐chains.
Yeast Phenylalanyl‐tRNA Synthetase
Authors: FASIOLO Franco , REMY Pierre , POUYET Jean , EBEL Jean‐Pierre 
Published: March 2005

The stoichiometries of phenylalanyl‐tRNA‐synthetase · tRNAPhe complexes were found close to 2, as determined by the following methods: zone centrifugation in sucrose gradient, Sephadex filtration, sedimentation boundary technique and fluorescence quenching of Y base of tRNAPhe. The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and tRNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for tRNAPhe. Under the experimental conditions used (pH 7.0 and 12°C) a dissociation constant of 3 × 10−8 M was calculated from the fluoresence quenching of tRNAPhe by the enzyme.tRNAPhe protects the enzyme against heat denaturation. A protection constant of 9 × 10−7 M was found at 55°C, pH 7.0. The presence of phenylalanine and ATP did modify the strength of interaction of the enzyme‐tRNA system. They induce, however, a synergistic effect of the protection afforded by tRNAPhe.The binding of phenylalanine was examined by equilibrium dialysis in presence of tRNAPhe. The stoichiometry obtained indicates two binding sites for phenylalanine. The two sites however do not possess the same affinity: the corresponding Kd values are 6 and 30μM. The presence of more than one active site is also manifested by the curvature of the double‐reciprocal plots of v versus [S] for phenylalanine, when measuring the initial rates of phenylalanyl‐tRNA formation. With regard to the site having the highest affinity, a 20‐fold higher Kd value was found, in the absence of tRNAPhe, when binding of the amino acid was followed by the phase partition technique.