bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence for Multiple Reactive Forms of Papain
Authors: POLGÁR László , HALÁSZ Piroska 
Published: March 2005

The pH‐dependence of the second‐order rate constants of acylation and alkylation reactions of the –SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different pKa values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (pKa= 3.6) and arginine derivatives (pKa= 4.3), the pKa differences can be attributed to electrostatic effects. However, the fact that a pKa of 3.25 was found with methyl and ethyl bromoacetate, and a pKa of 4.0 was obtained with bromoacetamide and a number of neutral substrates, is inconsistent with the theories put forth hitherto for the meaning of such pKa values, because they all consider only one reactive enzyme form. The different pKa values obtained here with neutral reactants are explained in terms of various reactive papain forms.The perturbation of pKa by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2‐mercaptoacetate, 2‐mercaptoethylamine, 2‐mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in pKa can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.
Cryoenzymologic Studies on Arginine Kinase: Solvent, Temperature and pH Effects on the Overall Reaction
Authors: TRAVERS Franck , BERTRAND Raoul , ROSEAU Gilbert , van THOAI Nguyen 
Published: March 2005

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substrates, kcat and pH profiles were investigated in detail.a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture.b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity.c) Ethylene glycol induces a large shift in the enzymic pKd) At ‐5 °C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate‐limiting step.The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.
Lipid‐Bound Oligosaccharides in Insects
Authors: QUESADA ALLUE Luis A. , BELOCOPITOW Enrique 
Published: March 2005

Membrane preparations from immature stages of the fruit fly Ceratitis capitata catalyze the transfer of mannose from GDP‐[14C]mannose into lipid‐linked oligosaccharides. These compounds behave as polyprenyl derivatives and their formation is stimulated by the addition of an acidic glyco‐lipid fraction isolated from insects.The mannose‐labeled oligosaccharides are attached to the poly‐isoprenol by a pyrophosphoryl linkage and can be released by mild acid hydrolysis. The trisaccharide lipid has been partially characterized. The results indicate that the compound is polyprenyl‐pyrophosphate‐N,N′‐diacetylchitobiose‐mannose.Incubation of dolichyl phosphate [14C]mannose or lower 14C‐labeled oligosaccharide lipids with unlabeled GDP‐mannose and the insect enzyme leads to the labeling of a higher lipid‐bound oligosaccharide.When UDP‐N‐acetyl[14C]glucosamine was incubated with insect membranes a 14C‐labeled chitobiosyl lipid was synthesized. If unlabeled GDP‐mannose was also present, the 14C label appeared in the trisaccharide and higher oligosaccharide lipids.Preliminary evidence indicates that the insect polyprenyl oligosaccharides described here might participate in glycoprotein biosynthesis.
Structure of Antigenic Determinants in the Amino‐Terminal Region of Bovine Fibrinogen Aα Chain
Authors: TANSWELL Paul , Hildegard Hildegard , TIMPL Rupert 
Published: March 2005

A radioimmunoassay was developed for peptide F‐CB1α from the amino end of bovine fibrinogen Aα chain, isolated after reduction and carboxymethylation of the multichain disulfide‐linked cyanogen bromide peptide F‐CB1. Seven out of twelve different rabbit antisera produced against fibrinogen, peptide F‐CB1 or Aα chain showed distinct binding to 125I‐labelled F‐CB1α. Thrombin cleavage of F‐CB1α yielded two fragments: fibrinopeptide A (residues 1‐19) and the carboxy‐terminal fragment Th2 (residues 20‐54). Antisera could be classified into three groups, according to whether they recognized antigenic determinants on fibrinopeptide A, on peptide Th2 or as they showed diminished reactions with both fragments. Only little or no cross‐reaction was observed with the amino‐terminal cyanogen bromide peptides of Bβ and γ chain.Proteolytic fragments of fibrinopeptide A were isolated and tested for inhibitory activity with two antisera. One antiserum contained antibodies binding selectively to the amino‐terminal sequence (residues 4‐11) and did not cross‐react with human fibrinopeptide A. Another antiserum showed a specific binding restricted to the carboxy‐terminal sequence (residues 11‐18) and cross‐reacted completely with human fibrinopeptide A. These results correlate well with the primary structures of the two fibrinopeptides. The antigenic activity of the peptide fragment Th2 was localized on a 15‐residue tryptic peptide derived from the central portion of the sequence. These and further data indicate that at least six different antigenic determinants are present in peptide F‐CB1α.
Production of NADPH in the Mannitol Cycle and Its Relation to Polyketide Formation in Alternaria alternata
Authors: HULT Karl , GATENBECK Sten 
Published: March 2005

The enzymes mannitol‐1‐phosphate dehydrogenase, mannitol‐1‐phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result:
The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.
Factors Modifying the Equilibrium between Activated and Non‐Activated Forms of Steroid‐Receptor Complexes
Authors: BAILLY Alain , SAVOURET Jean‐François , SALLAS Nicole , MILGROM Edwin 
Published: March 2005

Steroid‐receptor complexes formed in concentrated cytosol at low temperature, low ionic strength and neutral pH are unable to bind to nuclei. Various procedures are known to promote their ‘activation’. In the present work it is shown that an increase in temperature only enhances the rate of the reaction whereas no change in the equilibrium between activated and non‐activated complexes is observed. On the contrary an increase in ionic strength or pH, as well as a removal of a low‐molecular‐weight inhibitor, not only accelerate the reaction but also increase the concentration of activated complexes at equilibrium. Using two steroids differing 3‐fold in their affinity for the receptor, no difference was seen in the effect of the bound steroid on receptor activation.When combining various activation procedures it was observed that they acted independently of each other and additively. In all cases they retained their property of either modifying only the rate of the reaction or both its rate and equilibrium.Using changes in pH, it was also possible to induce shifts in the equilibrium between activated and non‐activated complexes. After activation at pH 6.5, a first equilibrium was attained. When the pH was increased to 8 the equilibrium was displaced towards higher concentrations of activated complexes. A lowering of the pH resulted in a reversal of steroid‐receptor complexes from the activated to the non‐activated state. To clearly establish that this was not due to irreversible damage of the receptor, which would render it unable to bind to nuclei, it was shown that the complexes which had reverted to the non‐activated to the non‐activated state were still susceptible to activation.Regulatory events may thus exist which, for a given level of hormone and receptor, modulate the concentration of activated steroid‐receptor complexes.
Adaptive Responses of Enzymes of Carbohydrate and Lipid Metabolism to Dietary Alteration in Genetically Obese Zucker Rats (fa/fa)
Authors: SPYDEVOLD Sverre O. , GREENBAUM A. Leslie , BAQUER Najma Zaheer , MCLEAN Patricia 
Published: March 2005

Measurements have been made of the activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis in liver and adipose tissue from genetically obese (fa/fa) rats and their lean litter mates (fa/‐). The effect of food restriction for a period of three weeks on the enzyme profile of liver and adipose tissue of the obese rat was also studied.
The most striking increases in enzyme activity in livers from obese rats were:(a) among enzymes of lipogenesis; ATP‐citrate lyase, acetyl‐CoA carboxylase, fatty acid synthetase, malate dehydrogenase (decarboxylating) and cytoplasmic glycerolphosphate dehydrogenase; (b) within the pentose phosphate pathway; glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase; (c) within the glycolytic pathway; glucokinase, pyruvate kinase and lactate dehydrogenase. All of these enzymes showed a significant increase in activity on the basis of U/g liver and U/mg DNA. In adipose tissue all the enzymes of lipogenesis, of the glycolytic route, of the oxidative segment of the pentose phosphate pathway and of the tricarboxylic acid cycle were increased when expressed as U/2 fat pads or as U/mg DNA.
The restriction of the food intake of obese rats to that consumed by their lean litter mates for periods of three weeks did not produce the expected adaptive decrease in enzymes of lipogenesis; in adipose tissue, only ATP‐citrate lyase and malate dehydrogenase (decarboxylating) showed a marked decrease; no significant change was found in adipose tissue or liver of the activities of acetyl‐CoA carboxylase and fatty acid synthetase, when expressed on a cell basis (U/mg DNA). The nonoxidative enzymes of the pentose phosphate pathway and enzymes involved in glycerogenesis (pyruvate carboxylase, malate dehydrogenase and phosphoenolpyruvate carboxylkinase) all increased in adipose tissue from limit‐fed obese rats.
The rate of conversion of specifically labelled glucose to [14C]O2 and 14C‐labelled lipid by pieces of adipose tissue and by liver slices was also measured. Insulin caused an increase in the conversion of [1‐14C]glucose to [14C]O2 and 14C‐labelled lipid in obese rats fed ad libitum, limit‐fed rats and in their lean litter mates.
The results are discussed in relation to the raised insulin and hypothyroid state of the obese rat. The effect of this altered hormonal status on the activity of cyclic nucleotide phosphodiesterases and cellular levels of adenosine 3′:5′ ‐monophosphate and guanosine 3′ :5′ ‐monophosphate in relation to the obese syndrome is considered.
Interaction between Membrane Functions and Protein Synthesis in Reticulocytes
Authors: WRESCHNER Daniel , MELLOUL Danielle , HERZBERG Max 
Published: March 2005

An inhibitor of protein synthesis has been isolated from reticulocyte membranes by solubilization with Triton X‐100; it has been purified using heat treatment, filtration on Amicon filters, DEAE‐cellulose ion‐exchange chromatography and Sephadex G‐75 gel chromatography. A final purification of 120‐fold was achieved.The purified inhibitor was found to be 95% homogenous when run on a dodecylsulfate/polyacrylamide gel system. Three independent methods were used to estimate the molecular weight of the purified inhibitor: Sephadex G‐75 gel chromatography, dodecylsulfate/polyacrylamide gel electrophoresis and sucrose gradient all confirmed that the purified inhibitor was a small molecule with a sedimentation coefficient of 0.7 S and a molecular weight ranging between 5000 and 8000.The purified inhibitor was shown to possess a specific endonucleolytic activity, degrading the 28‐S species of ribosomal RNA to species sedimenting between 10 and 14S. Due to its membrane localisation the name RNase M is proposed.The purified inhibitor's endonucleolytic activity was characterized with regard to its kinetics, concentration dependence, pH optimum and its requirements for divalent cations.Kinetics showed that RNase M retained its specificity after 60 min of incubation with the RNA substrate. Specificity was also demonstrated by incubating the polysomal RNA with high concentrations of purified enzyme. The pH optimum was found to be between pH 6 and pH 7, and the enzyme did not require divalent cations for its activity. Pancreatic RNase B used at a similar protein synthesis inhibitory concentration as the RNase M caused a complete breakdown of ribosomal RNA to oligonucleotides and mononucleotides.The possible biological significance of the purified inhibitor in regulating protein synthesis in the maturing reticulocyte is discussed.
The Complete Amino‐Acid Sequence of Histone H2B(3) from Sperm of the Sea Urchin Parechinus angulosus
Authors: STRICKLAND Marie , STRICKLAND Walter N. , BRANDT Wolf F. , VON HOLT Claus , WITTMANN‐LIEBOLD Brigitte , LEHMANN Arnold 
Published: March 2005

The primary structure of a third H2B histone isolated from sperm of the sea urchin Parechinus angulosus has been determined.H2B(3) consists of a polypeptide chain of the following 148 amino acid residues: Pro‐Arg‐Ser‐Pro‐Ala‐Lys‐Thr‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Ser‐Arg‐Lys‐Ala‐Ser‐Pro‐Lys‐Arg‐Gly‐Gly‐Lys‐Gly‐Ala‐Lys‐Arg‐Ala‐Gly‐Lys‐Gly‐Gly‐Arg‐Arg‐Arg‐Arg‐Val‐Val‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Glu‐Ser‐Tyr‐Gly‐Ile‐Tyr‐Ile‐Tyr‐Lys‐Val‐Leu‐Lys‐Gln‐Val‐His‐Pro‐Asp‐Thr‐Gly‐Ile‐Ser‐Ser‐Arg‐Ala‐Met‐Ser‐Val‐Met‐Asn‐Ser‐Phe‐Val‐Asn‐Asp‐Val‐Phe‐Glu‐Arg‐Ile‐Ala‐Ser‐Glu‐Ala‐Ser‐Arg‐Leu‐Thr‐Ser‐Ala‐Asn‐Arg‐Arg‐Ser‐Thr‐Val‐Ser‐Ser‐Arg‐Glu‐Ile‐Gln‐Thr‐Ala‐Val‐Arg‐Leu‐Leu‐Leu‐Pro‐Gly‐Glu‐Leu‐Ala‐Lys‐His‐Ala‐Val‐Ser‐Glu‐Gly‐Thr‐Lys‐Ala‐Val‐Thr‐Lys‐Tyr‐Thr‐Ser‐Arg.H2B(3)Parechinus closely resembles H2B(2)Parechinus but has one additional repeating pentapeptide in the amino‐terminal region and a serine replacing glycine at position 98.
Carboxylic and Polyethyleneimine‐Bound FAD Derivatives
Authors: ZAPPELLI Piergiorgio , PAPPA Rosario , ROSSODIVITA Antonio , RE Luciano 
Published: March 2005

Alkylation at N‐1 of the FAD adenine ring with 3,4‐epoxybutanoic acid gave 1‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Dimroth rearrangement of the latter afforded N6‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Coupling of the two carboxylic FAD derivatives to polyethyleneimine gave the corresponding soluble macromolecular FAD analogues.Fluorescence and circular dichroism determinations at neutral pH showed for the N‐1 carboxylic derivative an open conformation, while for the corresponding N6 derivative a stacked one as for FAD.All the synthesized derivatives have been shown to reactivate Aspergillus niger glucose oxidase and hog kidney d‐amino acid oxidase apoenzymes, affording holoenzymes whose activity relative to that of the corresponding natural holoenzymes ranged from 3 to 15% for the N‐1 derivatives and from 20 to 97% for the N6 derivatives.Investigations by ultraviolet difference spectra on the interaction with apo‐d‐amino acid oxidase showed substantial similarities between the N6 carboxylic derivative and FAD in the binding to the apoenzyme, while a completely different behaviour was observed for the N‐1 carboxylic derivative. The d‐amino acid oxidase apoenzyme, the holoenzyme and the glutaraldehyde‐treated holoenzyme were entrapped each in cellulose triacetate fibres together with the polyethyleneimine N6 derivative and beef liver catalase; the operational stability of the three recycling systems with dl‐alanine as substrate was shown to be respectively 6, 5 and 12 times higher than the one found for the entrapped natural holoenzyme system.
Biochemical Characterization of the Restriction‐Modification System of Bacillus sphaericus
Authors: KONCZ Csaba , KISS Antal , VENETIANER Pál 
Published: March 2005

A type II restriction endonuclease (endo R ·Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported.The enzyme also cleaves single‐stranded DNA, albeit at a slower rate. It seems likely that single‐stranded DNA is cleaved at the same sequences as double‐stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M ·Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S‐adenosyl‐methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R ·Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG‐dG‐dC‐dC) as the nuclease.
Purification and Characterization of an Endonuclease from Micrococcus luteus that Acts on Depurinated and Carcinogen‐Modified DNA
Authors: HECHT Reinhardt , THIELMANN Heinz Walter 
Published: March 2005

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500‐fold from Micrococcus luteus. In this purification, separation from the pyrimidine‐dimer‐specific ultraviolet‐endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg27plus;, but 5–30 mM Mg27plus; produces a five‐fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single‐strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N‐alkyl‐N‐nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, β‐propiolactone, 7‐bromomethyl‐benz[a]anthracene, N‐acetoxy‐2‐acetylaminofluorene, and 7,12‐dimethyl‐benz[a]anthracene‐5,6‐oxide also becomes susceptible to enzymic action.The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed‐circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel electrophoresis. Even depurinated or carcinogen‐modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms.A comparison of the number of enzyme‐catalyzed single‐strand breaks with the number of alkali‐labile (i.e. apurinic) sites in carcinogen‐modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet‐irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme‐catalyzed single‐strand breaks found in ultraviolet‐irradiated DNA was five times the number of alkali‐labile sites present suggesting that minor photoproducts, possibly 5,6‐saturated pyrimidine residues, were recognized in addition to apurinic sites.
Native and Latent Forms of Liver Phosphorylase Phosphatase
Authors: LALOUX Monique , STALMANS Willy , HERS Henri‐Géry 
Published: March 2005

The directly measurable (native) phosphorylase phosphatase present in a fresh mouse liver extract is bound to particulate glycogen and is not inhibited by heat‐stable inhibitors. Treatment of the extract with trypsin or ethanol at room temperature caused a more than 10‐fold increase in phosphorylase phosphatase activity. This increased activity stems from the activation of completely inactive (latent) enzyme, the major part of which is present in the high‐speed supernatant. The trypsin‐revealed activity can be completely blocked by heat‐stable inhibitors. Treatment of the animal with glucocorticoids increases, and fasting decreases the activity of the native phosphorylase phosphatase. The level of latent enzyme, however, is unaffected by these treatments. The major portion of synthase phosphatase in the fresh liver extract is bound to glycogen. This enzyme is inhibited by the heat‐stable inhibitor‐2 and inactivated by trypsin or ethanol as well as by several treatments that have little effect on phosphorylase phosphatase.Upon DEAE‐cellulose chromatography at 0°C of a fresh liver extract, phosphorylase phosphatase and synthase phosphatase were resolved as separate, single peaks. If the preparation was not kept at 0°C during the entire procedure, two peaks of each enzyme were observed. Under these conditions the first peak of phosphorylase phosphatase and of synthase phosphatase coincided. From these findings it is concluded that synthase phosphatase and phosphorylase phosphatase, in their native form, are distinct enzymes.