bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells
Authors: GROEN Albert K. , SIPS Herman J. , VERVOORN Richard C. , TAGER Joseph M. 
Published: March 2005

The catabolism of alanine in isolated rat liver cells was studied using a perifusion system in order to titrate the cells with different concentrations of alanine.The Concentrations of the components of the alanine aminotransferase reaction in the cytosolic and mitochondrial compartments were measured after digitonin fractionation of samples of the cell suspension taken during each steady state. Comparison of the mass‐action ratios with the equilibrium constant indicated that the cytosolic enzyme is poised towards pyruvate formation at all concentrations of alanine used, whereas the mitochondrial enzyme is always poised towards alanine formation. The calculated flux through cytosolic alanine aminotransferase, using literature data for the kinetic parameters of the enzyme and experimental data for the concentration of metabolites, was in close agreement with the measured carbon flux. It is concluded that the transamination step in alanine catabolism occurs in the cytosol.The concentration of the components of the glutamate dehydrogenase and 3‐hydroxybutyrate dehydrogenase reactions in the mitochondrial compartment during each steady state were also measured. The ΔG of the combined reactions did not change appreciably on increasing the extracellular alanine concentration to 0.6 mM. At higher alanine concentrations ΔG became slightly more negative. As the extracellular Concentration of alanine was increased, the concentration of glutamate in both the cytosolic and the mitochondrial compartments increased. However, the cytosolic alanine concentration was always lower than that in the extracellular compartment even at the highest concentration of alanine (6 mM) infused. When increasing concentrations of pyruvate were infused together with a fixed concentration of alanine (0.6 mM), the cytosolic concentration of alanine increased to levels even higher than that in the perifusate. However, there was no significant effect in the rate of formation of nitrogenous products.It is concluded that the transport of alanine across the plasma membrane is the only rate‐controlling step in alanine catabolism under the conditions used.
Purification and Characterization of a Mannose/Glucose‐Specific Lectin from Vicia cracca
Authors: BAUMANN Christian M. , STROSBERG A. Donny , RÜDIGER Harold 
Published: March 2005

The seeds of Vicia cracca are known to contain two lectins: an N‐acetylgalactosamine‐binding lectin, which reacts specifically with human erythrocytes of blood group A, and a mannose/glucose‐binding lectin unspecific towards human blood groups. The second lectin is the object of the present work. Purification was achieved by affinity chromatography on Sephadex G‐100. The native lectin has a molecular weight of 44000. It is composed of two small (Mr 5700) and two large subunits (Mr 17500). Binding of sugars to the lectin was assayed by equilibrium dialysis and spectrophotometrically; the association constants for glucose are 1.38×103 l/mol and 2.5×102 l/mol respectively.The primary structure of the small subunit was determined by analyses of the whole chain, of tryptic and CNBr peptides. It was shown to consist of 53 amino acid residues. The mannose/glucose‐binding lectin from Vicia cracca is highly homologous to the lectins from Vicia sativa, Vicia faba, Pisum sativum and Lens culinaris. There is, however, only limited homology to the N‐acetylgalactosamine‐specific lectin from Vicia cracca.
Studies on Prophospholipase A2 and Its Enzyme from Human Pancreatic Juice
Authors: GRATAROLI Renerq;e , DIJKMAN Ruuid , DUTILH Christian E. , VAN DER OUDERAA Frans , DE HAAS Gerard H. , FIGARELLA Catherine 
Published: March 2005

Upon tryptic activation of pure human prophospholipase A2, a heptapeptide is released from the N‐terminal part of the protein yielding active phospholipase A2 (EC 3.1.1.4). Both the kinetics of the activation process and the amino acid sequence of the activation peptide strongly resemble those of pancreatic zymogens of other mammalian sources.The kinetic properties of human phospholipase A2 and its zymogen are compared with those of the corresponding porcine enzyme using substrates present as micelles, molecular dispersed solutions or as monomolecular surface films. The most obvious difference between the human and porcine phospholipase A2 is the low enzyme activity of the former protein at pH 8.0 as compared to pH 6.0, both against micellar and monomeric substrates. Neither the Ca2+ binding properties nor the inhibition kinetics of the human enzyme using haloketones can easily explain this different pH optimum.The sequence analysis of the N‐terminal region of the first 40 residues is reported.
Incorporation of N‐Ethylmaleimide into the Membrane‐Bound ADP/ATP Translocator
Authors: AQUILA Heinrich , EIERMANN Wolfgang , KLINGENBERG Martin 
Published: March 2005

The incorporation of N‐ethylmaleimide into the 30000‐Mr component of beef‐heart mitochondria has been studied as a function of various ligands to the ADP/ATP carrier and the isolation of the N‐ethylmaleimide‐labeled protein is reported.

The incorporation of N‐ethylmaleimide into the 30000‐Mr component is specifically stimulated by ADP and ATP. Thus by differential incorporation of N‐ethylmaleimide, the 30000‐Mr component is preferentially labeled.
Addition of carboxyatractylate inhibits, whereas bongkrekate tolerates, the incorporation of N‐ethyl‐maleimide.
After solubilization by Triton the purification of N‐ethylnialeimide‐labeled protein is facilitated in the presence of bongkrekate but not of carboxyatractylate, in agreement with the postulated existence of only a bongkrekate‐ N‐ethylmaleimideprotein complex. The labeled protein was purified to homogeneity on hydroxyapatite in Triton and subsequently, after denaturation in dodecylsulfate, on Sepharose 6B.
The identity of the isolated labeled protein with the formerly isolated bongkrekate‐protein or carboxy‐ atractylate‐protein complexes is confirmed by the isoelectric point and amino acid composition.
Two moles of N‐ethylmaleimide must be incorporated into the 30000‐Mr component in order to inhibit fully the binding of one mole carboxyatractylate. This corresponds to one ‐SH group per unit.
The Reactivity of ‐SH Groups in the ADP/ATP Carrier Isolated from Beef Heart Mitochondria
Authors: AQUILA Heinrich , KLINGENBERG Martin 
Published: March 2005

The emergence of the reactivity of ‐SH groups associated with conformational changes has been studied on the ADP/ATP carrier, as isolated in three different inhibitor‐protein complexes.

The bongkrekate‐protein complex incorporates approximately one molecule more of N‐ethylmaleimide than the carboxyatractylate‐protein complex. After extensive denaturation by dodecylsulfate in urea, both inhibitor complexes exhibit four reactive ‐SH groups per subunit. Thus one out of four ‐SH groups per subunit has been unmasked in the bongkrekate‐protein complex.
The interconversion from the bongkrekate‐protein complex to the carboxyatractylate‐protein complex is inhibited after the ‐SH groups have been blocked.
The protein complex, isolated with the more easily dissociable atractylate, is used to demonstrate, by the emergence of the ‐SH groups, the transition into the m‐state. This transition is specifically catalyzed by ADP and ATP.
Using 2,2′‐dinitro‐5,5′‐dithiodibenzoate, the appearance of the ‐ SH groups on transition from the c‐state to the m‐state can be followed spectrophotometrically. The specificity for the catalyzing nucleotides is identical with that for transport. The Km for ADP and ATP is in the range of 1 μM.

In conclusion, the thiol groups of the isolated ADP/ATP carrier behave as in the mitochondria1 membrane. The unmasking of ‐SH groups is in full accordance with the concept of two conformational states (c and m)
Heterogeneity of Binding Sites for Adsorptive Pinocytosis of Simple Proteins by Rat Yolk Sacs
Authors: LIVESEY Geoffrey , WILLIAMS Kenneth E. 
Published: March 2005

When rat yolk sacs were incubated in serum‐free medium 199, the 251I‐labelled forms of both formaldehyde‐treated bovine serum albumin and ribonuclease were captured far more rapidly than 125I‐labelled polyvinylpyrrolidone. Extensive adsorption of these proteins to the plasma membrane is the main cause of this effect.
Quantitative analysis of the adsorptive‐phase pinocytosis of these two proteins showed curved Hofstee plots, suggesting either the presence of multiple classes of binding site on the surface of pinocytically‐active yolk‐sac cells or a single class of binding site that exhibits negative cooperativity in the binding of these proteins. The values of Km were similar, ranging over 0.6‐ 11.8 μM for 125I‐labelled ribonuclease and over 0.31 ‐4.7 μM for formaldehyde‐treated 125I‐labelled albumin.
Competitive uptake studies, in which tracer amounts of each of the 125I‐labelled proteins were ingested from serum‐free medium containing a higher concentration of one of a series of unlabelled proteins, revealed marked differences between the two radiolabelled proteins. These findings suggest that formaldehyde‐denatured albumin is captured by binding to hydrophobic binding sites on the plasma membrane whereas ribonuclease is captured by binding to negatively charged sites.
Ribitol Dehydrogenase Messenger RNA from an Enzyme Superproducer Strain of Klebsiella aerogenes
Authors: BAHRAMIAN Mohammad B. , HARTLEY Brian S. , LOVINY T. 
Published: March 2005

Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti‐(ribitol dehydrogenase) and immunoprecipitating with sheep anti‐(rabbit IgG).
The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35–40‐times more efficiently than RNA from coliphages MS2 or Qβ, to give ribitol dehydrogenase as sole major product.
Its size distribution shows a major band of 1500 nucleotides plus fragments 400–1400 nucleotides, with only traces of size 2400–3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D‐ribulokinase.
Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a ‘switch’ point in mid‐exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA.
This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.
Carp Insulin: Amino Acid Sequence, Biological Activity and Structural Properties
Authors: MAKOWER Alexander , DETTMER Rudolf , RAPOPORT Tom A. , KNOSPE Siegfried , BEHLKE Joachim , PREHN Siegfried , FRANKE Peter , ETZOLD Gerhard , ROSENTHAL Sinaida 
Published: March 2005

The amino acid sequence of insulin of carp (Cyprinus carpio) has been determined and correlated with its biological activity in a fat‐cell test and its structural properties as measured by circular dichroism and sedimentation analysis.The amino acid sequence of carp insulin displays some unusual features: the B chain is longer at the N ter‐ minus by two residues as compared with mammalian insulins and there are substitutions of the charged residues, found in most insulins at positions B21 and B22, by proline and threonine respectively. On the other hand, all amino acid residues essential for biological activity and for the association of insulin monomers are the same in carp insulin. Accordingly, the half‐maximal response in a fat‐cell test is reached with carp insulin at concentrations which are only three times higher than with porcine insulin and the maximal response is the same. The circular dichroism spectrum of carp insulin resembles greatly that of bovine insulin indicating that it has a similar spatial structure. Despite amino acid substitutions in the dimer‐dimer contact region, carp insulin is able to form hexamers.
Citrate Synthesis in Intact Rat‐Liver Mitochondria Is Irreversible
Authors: GREKSAK Miloslav , LOPES‐CARDOZO Matthijs , VAN DEN BERGH Simon G. 
Published: March 2005

Rat‐liver mitochondria were incubated with [1,5‐14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion‐exchange chromatography. Incorporation of 14C into acetyl‐l‐carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol. mg−1. min−1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg−1 . min−1.We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.
Generation of Prothrombin‐Converting Activity and the Exposure of Phosphatidylserine at the Outer Surface of Platelets
Authors: BEVERS Edouard M. , COMFURIUS Paul , VAN RIJN Jan L. M. L. , HEMKER H. Coenraad , Robert F. A ZWAAL  
Published: March 2005

In the present paper we compare prothrombin‐converting activities of platelets non‐activated, or activated by collagen, thrombin or collagen plus thrombin in the absence and presence of added factor Va. In all experiments described, the rate of thrombin formation for platelets activated by the combined action of collagen and thrombin is greater than that of platelets stimulated by collagen or thrombin alone. The presence of added factor Va enhanced the rate of thrombin formation in all cases, but the higher activity observed with platelets stimulated by collagen plus thrombin remains. When platelets are activated by collagen plus thrombin in the presence factor Xa and prothrombin, a lag period of approximately 10 min is observed before the rate of thrombin formation reaches a steady state. Addition of an excess of factor Va in this experiment reduces the lag time to 3 min. This lag period is interpreted as the time required to generate extra binding sites for the prothrombinase complex at the platelet surface. These extra sites explain the difference in thrombin formation rate between these platelets and platelets activated by either collagen or thrombin only.The exposure of phospholipids at the platelet outer surface was studied with phospholipases after various activations of the platelets. It is demonstrated that activation by collagen plus thrombin is accompanied by increased susceptibility of platelet phospholipids towards phospholipase A2. Among these degradable phospho‐ lipids are 25% of the phosphatidylserine and 30% of the phosphatidylethanolamine. On the other hand, little no phosphatidylserine is exposed at the membrane exterior of thrombin‐treated or control platelets. We propose that the exposure of phosphatidylserine at the outer surface of platelets activated with thrombin plus collagen essential for the rate enhancement of thrombin formation observed under these conditions. The possibility of transbilayer movement of phospholipids in the platelet membrane as a result of the activation process will be discussed.
pH and Temperature Dependences of Thermolysin Catalysis
Authors: KUNUGI Shigeru , HIROHARA Hideo , ISE Norio 
Published: March 2005

The pH dependences of kCat/Km, kcat and Km of thermolysin‐catalyzed hydrolysis of N‐furylacryloylglycyl‐l‐leucinamide (Fua‐Gly‐LeuNH2) and N‐furylacryloylglycyl‐l‐phenylalaninamide (Fua‐Gly‐PheNHz) were in‐ vestigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell‐shaped curve with pKa1= 5.0 and pKa2= 8.25, at 25°C. Both kcat and Km increased with pH at lower pH and took larger values for Fua‐Gly‐LeuNHz than for Fua‐Gly‐PheNHz at 25°C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy‐L‐phenylalanine and L‐phenylalanyl‐I–leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH.Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non‐linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated AH values of deprotonation for pKal and pKa1 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKal is too large to be assigned to the carboxylic group of Glu‐143, in contrast to the generally accepted view.A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc‐coordinated water
Dimerization of Deoxyribonuclease I, Lysozyme and Papain
Authors: SORRENTINO Salvatore , YAKOVLEV Gennady I. , LlBONATI Massimo 
Published: March 2005

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskočil, J., and Libonati, M. (1980) Biochim. Bioplhys. Acta, 609, 40–52]. To see whether this phenomenon could be common to other enzyme‐substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease. hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg‐white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength.Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross‐linkage with dimethyl suberimidate.The main results of the investigation, similar to those obtained with ribonuclease A, are the following.1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one.2.The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme.A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non‐specific interactions with polyelectrolytic substrates.
The Chemical Structure of Lipid A
Authors: WOLLENWEBER Horst‐Werner , BROADY Kevin W. , LUDERITZ Otto , RIETSCHEL Ernst Th 
Published: March 2005

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated β1,6‐linked D‐glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, D‐3‐hydroxytetradecanoic and D‐3‐O‐(tetradecanoyl)‐tetradecanoic acid in ester linkage and two of D‐3‐hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopoly‐ saccharide with alkali at elevated temperature leads, through a β‐elimination reaction, to the generation of amice‐ bound δ2‐tetradecenoic acid. This suggested that the 3‐hydroxyl group of amide‐bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas‐liquid chromatography/mass spectrometry the methyl esters of 3‐O‐(dodecanoyl)‐tetradecanoic and 3‐O‐(hexadecanoyl)‐tetradecanoic acid were identified. This shows that in lipid A amide‐linked 3‐hydroxytetradecanoic acid residues are 3–0‐acylated by dodecanoic and hexadecanoic acid, respectively.Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D‐3‐hydroxytetra‐ decanoyl residues, two being present as esters and two as amides. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3‐hydroxytet radecanoyl residues: specifically, tetradecanoic acid substitutes ester‐bound and dodecanoic and hexadecanoic acid amide‐bound 3‐hydroxytetradecanoic acid.
Carbohydrate Complexity of the Mouse Thymocyte Thy‐1 Glycoprotein as Demonstrated by Lectin Affinity and Isoelectric Focusing
Authors: CARLSSON Sven , STIGBRAND Torgny 
Published: March 2005

The Thy‐I glycoprotein of mouse thymocytes was analysed with regard to the properties of its Carbohydrate part. Of the different lectins tested, partial binding of Thy‐1 from membrane extracts was found to Lens culinuris agglutinin (approximately 50%) and wheat germ agglutinin (approximately 25%). In contrast, concanavalin A bound all Thy‐I. The results indicate that there is considerable microheterogeneity in the oligosaccaride chains within the Thy‐I population.Thy‐I was purified from thymocyte membrane extracts by concanavalin A affinity chromatography and gel filtration. The purified preparation revealed, on analysis by isoelectric focusing, at least six charge variants (isoelectric points ranging from approximately 5–9). These variants were isolated, treated with neuraminidase and tested for lectin binding and isoelectric point. It was shown that the charge heterogeneity was due to different amounts of sialic acid; even the most basic form contained at least one sialic acid residue. Furthermore, it was concluded that the degree of sialylation was not correlated to the lectin‐binding properties.Analysis of Thy‐1 from surface 125I‐labelled thymocytes showed that the purified preparation was representative in its complexity of the Thy‐I glycoprotein exposed on the surface membrane. The possible biological implications of the findings are discussed.