bluuebunny/crossref_metadata_2025_split huggingface dataset:
Relationship between the Pharmacological Action of Crotoxin and Its Phospholipase Activity
Authors: MARLAS Guy , BON Cassian 
Published: March 2005

Crotoxin, the major neurotoxin from Crotalus durissus terrificus venom, blocks the postsynaptic response of Electrophorus electroplaque to bath‐applied carbamoylcholine. Crotoxin is composed of two non‐covalently linked subunits: an acidic non‐enzymatic component, A, and a basic phospholipase component, B. According to our previous studies, component A restricts the binding of component B to high‐affinity sites which occur in Torpedo membrane fragments in approximatively equivalent amount to the cholinergic receptor sites. We demonstrate here that the catalytic activity of component B is actually required for the postsynaptic blocking action of the toxin.Irreversible inactivation of component B was achieved by alkylation with p‐bromophenylacyl bromide of a histidine residue in the catalytic site. Although the native crotoxin complex was insensitive to the inhibitor, suggesting that the catalytic center is protected by component A, we obtained an enzymatically inactive crotoxin by recombination of the alkylated component B with component A. This modified crotoxin was devoid of enzymic activity, toxicity and pharmacological activity on the electroplaque.The phospholipase activity of component B may be reversibly inhibited by replacing Ca2+ with Sr2+ ions in the physiological solution. This allowed us to identify several steps in the action of the toxin: a quasi‐irreversible binding step which occurs either in the presence of Ca2+ or Sr2+ ions, and a catalytic step which requires Ca2+ ions and is responsible for the irreversible blockade of the electroplaque response to cholinergic agonists.Finally, we examined the effect of various toxic and non‐toxic phospholipases on the electroplaque response. The data exclude that the postsynaptic blockade results from indiscriminate hydrolysis of phospholipidb. They rather suggest that the pharmacological efficiency of crotoxin is due to the fact that component B exerts its catalytic activity at critical postsynaptic sites. These conclusions are in excellent agreement with the explanation of the synergistic effect of the two subunits which we derived from our binding data. The postsynaptic sensitive sites for crotoxin action might be closely related to the acetylcholine receptor sites.
Direct Measurement of Proton Transfer as a Probing Reaction for the Microenvironment of the Apomyoglobin Heme‐Binding Site
Authors: GUTMAN Menachem , NACHLIEL Esther , HUPPERT Dan 
Published: March 2005

Aromatic alcohols fluoresce at different wavelengths in their neutral (øOH*) and anionic (øO*) excited states. Consequently, time‐resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state.8‐Hydroxypyrene 1,3,6‐trisulfonate (pK°= 7.7, pK*= 0.5) is bound to apomyoglobin with a stoichiometry of 1 : 1. In the bound state its neutral form fluorescence increase 20‐fold.The binding affinity is pH‐dependent. Two protonatable groups, with pK= 6.5, participate in the stabilization of the negatively charged ligand in the binding site.The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin.Time‐resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin.The rate of proton dissociation is slowed to 7 %, of the rate measured for the free ligand. Such a slow disso‐ ciation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637‐642]. The water activity in the site is equivalent to a (H2O) = 0.67.
Somatic Antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander S. , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M. 
Published: March 2005

Structural and immunochemical studies have been performed on the 0‐specific antigens of two serologically related types of Pseudomonas aeruginosa 0 : 2a,b and 0 : 2a,c (Lanyi's classification). The 0‐specific polysaccharide chains of the two serotypes were shown to be acidic polysaccharides composed of repeating trisaccharide units consisting of l‐rhamnose, N‐acetyl‐d‐quinovosamine and N‐acetyl‐L‐galactosaminuronic acid residues. Based on 1 H and 13C nuclear magnetic resonance spectroscopy, data of methylation analysis and selective solvolysis with hydrogen fluoride, the repeating unit of the 0 : 2a,c 0‐specific polysaccharide was assigned the following structure →4)l‐GalNAcA(α1→ 3)d QuiNAc(αl→ 3)LRha(α1, where GalNAcA =N‐acetylgalactosaminuronic acid, QuiNAc= acetylquinovosamine and Rha = rhamnose.The 0:2a,b 0‐specific polysaccharide had the same structure of the trisaccharide repeating unit but was distinguished by the presence of 0‐acetyl groups on 70–80% of the rhamnose residues in position 2. The 0‐acetyl groups were located both by methylation with methyltrifluoromethane sulphonate and by comparison of the 13C nuclear magnetic resonance spectra of the native and 0‐deacetylated polysaccharides.Serological studies revealed the determinant role of the 0‐acetyl groups in the 0‐antigen 0 : 2a,b and suggested the 0‐factor 2b to be related to the 2‐0‐acetyl‐l‐rhamnose residue, whereas the 0‐factor 2c was probably deter‐ mined by the nonacetylated rhamnose residue. The dominant moiety of the determinant 2a common to the two antigens was obviously presented by the N‐acetyl‐l‐galactosaminuronic acid residue.
Direct Evidence that Cholesterol Ester Hydrolase from Adrenal Cortex is the Same Enzyme as Hormone‐Sensitive Lipase from Adipose Tissue
Authors: COOK Kenneth G. , YEAMAN Stephen J. , STRALFORS Peter , FREDRIKSON Gudrun , BELFRAGE Per 
Published: March 2005

Several properties of the cytosolic cholesterol ester hydrolase from bovine adrenal cortex were investigated and those properties were compared directly with those of the well‐characterised hormone‐sensitive lipase, the rate‐limiting enzyme in adipose tissue lipolysis. Properties examined included : (a) activity against different substrates; (b) susceptibility to inhibition by NaF, Hg2+ ions and diisopropyl fluorophosphonate; (c) subunit molecular weight as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; (d) ability to serve as a substrate for cyclic AMP‐dependent protein kinase; (e) effect of phosphorylation on enzyme activity: and (f) degradation pattern of polypeptides following limited proteolysis.In all respects the two enzymes exhibited essentially identical characteristics. It is therefore concluded that the same protein, or two very similar proteins, catalyses the hydrolysis of cholesterol esters in adrenal cortex and lipolysis in adipose tissue.The implication of this finding is discussed in relation to the hormonal control of steroidogenesis in adrenal cortex and of lipolysis in adipose tissue.
Restoration of NADH Oxidation with Menaquinones and Menaquinone Analogues in Membrane Vesicles from the Menaquinone‐Deficient Bacillus subtilis aroD
Authors: BERGSMA Jack , MEIHUIZEN Kor E. , Van OEVEREN Wim , KONINGS Wil N. 
Published: March 2005

Membrane vesicles from the menaquinone‐deficient Bacillus subtilis aroD oxidize NADH at a low rate. NADH oxidation can be restored by the addition of slightly water‐soluble menaquinone and ubiquinone analogues up to saturation levels. These saturation levels differ for the different quinone analogues tested from 95 (1,4‐benzo‐ quinone) to 5316 (5‐hydroxy‐1,4‐naphthoquinone, juglon) nmol NADH × min−1× mg membrane protein−1 NADH oxidation in membrane vesicles from B. subtilis aroD restored with water‐soluble quinone analogues supplies the energy for l‐glutamate uptake. Like NADH oxidation the initial rate of l‐glutamate transport increases up to saturation levels. The highest initial rates of l‐glutamate uptake are observed after restoration with quinone analogues with a relatively low standard redox potential.Functional reconstitution with natural, water‐insoluble, quinones can be achieved effectively by mixing quinone‐containing liposomes with membrane vesicles from B. subtilis aroD and subsequent freezing of the mixture in liquid nitrogen. The rate of NADH oxidation increased with the amount of menaquinone incorporated in the vesicles up to saturation levels. NADH oxidation via these menaquinones also supplies the energy for l‐glutamate uptake. The highest uptake rates can be obtained with menaquinone‐1 and menaquinone‐2. On the basis of efficiencies (mol NADH oxidized/mol l‐glutamate transported) menaquinones and mena‐ quinone analogues can be divided in two classes.To class 1 belong the menaquinone analogues, menaquinone‐5 and menaquinone‐8. These compounds restore NADH oxidation with low levels of energy transduction. Efficiencies are observed which are comparable with the efficiency observed in membrane vesicles from B. subtilis W23 (120‐140) which contain the natural mena‐ quinone‐7.To class 2 belong menaquinone‐I and menaquinone‐2, which restore NADH oxidation with high levels of energy transduction. Efficiencies are observed which are in the same range as observed with phenazine methosulphate (9‐13).A model is proposed in which class 1 compounds feed in electrons from the outside from NADH to the Q‐cycle of the respiratory chain. Class 2 compounds donate electrons to the respiratory chain after cytochrome c and before cytochrome α‐601.
Inhibition of Pyruvate Dehydrogenase during the Metabolism of Glutamine and Proline in Hemoglobin‐Free Perfused Rat Liver
Authors: HÄUSSINGER Dieter , GEROK Wolfgang , SIES Helmut 
Published: March 2005

A shift of pyruvate dehydrogenase from the active (dephospho) towards the inactive (phospho) form results from the metabolism of added glutamine in isolated perfused rat liver. This is evidenced by a decreased release of 14CO2 from [1‐14C]pyruvate and by direct measurement of enzyme activity in the freeze‐stopped liver tissue. Half‐maximal glutamine concentration for the inhibition of pyruvate oxidation is about 1 mM, i.e. within the physiological range. The effect is reversible and exhibits a time course similar to that of glutamine metabolism as measured by the production of ammonia and urea. The effect is independent of the mode of glutaminase activation, either by ammonium ions or glucagon or by so‐called ‘self‐activation’.
When oxoglutarate, glutamate and ammonia are generated intracellularly from proline, there is also an inhibition of 14CO2 release from [1‐14C]pyruvate. Proline metabolism is stimulated about twofold by addition of glucagon. This is accompanied by a further decrease of pyruvate dehydrogenase activity, whereas addition of glucagon alone has no effect.
There is good agreement between 14CO2 production from [1‐14C]glutamine and oxoglutarate production as calculated from nitrogen balance measurements. This indicates that oxoglutarate formed from glutamine is predominantly oxidized by oxoglutarate dehydrogenase. Furthermore, there is a close correlation between oxidation of oxoglutarate derived from glutamine or proline and the degree of inhibition of pyruvate dehydrogenase. This correlation holds also when oxoglutarate production from glutamine or proline is increased by ammonium ions or glucagon. Thus, it is concluded that increased flux through oxoglutarate dehydrogenase is accompanied by a decreased flux through pyruvate dehydrogenase in rat liver.
Interference of Ligands on the Phenylalanyl‐tRNA Synthetase from Yeast
Authors: THIEBE Rainer 
Published: March 2005

The present paper reports a study of the mutual interactions between the substrates, the intermediate, and the products of the aminoacylation reaction, when bound to the phenylalanyl‐tRNA synthetase from yeast. The following conclusions can be drawn.

tRNAPhe displaces Phe‐tRNAPhe from the synthetase by lowering the affinity of the enzyme for the amino‐acylated tRNA.
Phe‐tRNAPhe and Phe‐AMP compete for the catalytically active site of the enzyme.
Chemically synthesized Phe‐AMP, when added to the synthetase, primarily forms a low‐affinity complex with the enzyme. The transformation of this complex into the high‐affinity catalytic complex is a very slow process.

These findings confirm a previous study, based on steady‐state kinetics. A schematic representation of the aminoacylation process is given. It summarizes the present and previous results and illustrates a rather complex ‘flip‐flop’ mechanism.
Mechanism of Synthesis of Adenosine(5′)tetraphospho(5′)adenosine (AppppA) by Aminoacyl‐tRNA Synthetases
Authors: GOERLICH Ottmar , FOECKLER Renate , HOLLER Eggehard 
Published: March 2005

Synthesis of radioactive adenosine(5′)tetraphospho(5′)adenosine in the presence of aminoacyl‐tRNA synthetases of procaryotes or eucaryotes was measured by thin‐layer chromatography on poly(ethyleneimine)‐cellulose sheets. Catalysis by the majority of aminoacyl‐tRNA synthetases was observed. They were classified into a fast group (specificity for Lys, Phe, His), a slow group (Ser, Ile, Leu, Asp, Tyr, Val) and a non‐catalytic group (Trp, Arg). The presence of the cognate amino acid is required.The mechanism was verified by direct synthesis of the tetraphosphate from preformed aminoacyladenylate and by its reverse reaction from chemically synthesized adenosine(5′)tetraphospho(5′)adenosine. Essentials are as follows. (1) Aminoacyladenylate is synthesized from a first molecule of MgATP. (2) A second ATP adds as substrate, Michaelis‐Menten constants being 11 mM and 50 mM in the case of Escherichia coli lysine‐specific and phenylalanine‐specific enzymes, respectively; these values are two orders of magnitude higher than for the first ATP. (3) This is followed by the synthesis of adenosine(5′)tetraphospho(5′)adenosine from adenylate and the second ATP; the reaction is rate‐determining in catalysis, rate constants being 0.5 s−1 and 0.26 s−1 respectively. (4) The equilibrium constant, K∼ 0.02, does not favour the synthesis of the tetraphosphate from ATP; removal of accumulating pyrophosphate by the action of added inorganic pyrophosphatase increases yields substantially. (5) The presence of Mg2+ ions is essential; in a second role, they can strongly activate the catalytic reaction. (6) Zn2+ is a more potent activator, although it is not essential; its effect was to decrease the value of the Michaelis‐Menten constant and to increase that of the catalytic rate constant; activation was observed only for a minority of aminoacyl‐tRNA synthetases (Phe, Lys).The synthesis of adenosine(5′)tetraphospho(5′)adenosine and of adenosine(5′)triphospho(5′)adenosine and its reverse are discussed with regard to possible biological functions.
Presence of an Amino Acid Oxidase in Photosystem II of Anacystis nidulans
Authors: PISTORIUS Elfriede K. , VOSS Helga 
Published: March 2005

Membrane fractions from the blue‐green algae Anacystis nidulans can catalyze O2 evolution with ferricyanide as acceptor in the light and the dark oxidation of l‐arginine. The two reactions are both inhibited by o‐phenanthroline, but they respond to certain trivalent, divalent and monovalent cations (like e.g. La3+ or Ca2+ or K+) in different ways. These metals stimulate the Hill reaction with ferricyanide as acceptor, but totally inhibit l‐arginine oxidation. The order of effectiveness is M3+ > M2+ > M+. The effect of e.g. Ca2+ on the dark oxidation of l‐arginine mirrors the effect of Ca2+ on the light‐driven O2 evolution with ferricyanide, as though these two activities are mutually exclusive, i.e. one process is inhibited to the extent that the other is activated. The enzyme responsible for the dark oxidation of l‐arginine has been purified from A. nidulans and shown to be a flavo‐enzyme [Pistorius, E. K. and Voss, H. (1980) Biochim. Biophys. Acta, 611, 227–240].Active photosystem II particles have also been isolated from Anacystis membrane fractions. They can catalyze 2,6‐dichloroindophenol reduction with diphenylcarbazide as donor in the light. They retain l‐arginine oxidation activity in the presence of EDTA. Both reactions are inhibited by o‐phenanthroline. Moreover, it could be shown that one of the protein bands which has been shown to be characteristic for photosystem II particles [Koenig, F. and Vernon, L. P. (1981) Z. Naturforsch. C36, 295–304], co‐chromatographs with the purified l‐amino‐acid oxidase on SDS gels. An antibody prepared to the purified l‐amino‐acid oxidase also gave a precipitin line with detergent‐treated photosystem II particles.The experiments show that the l‐amino‐acid oxidase is part of photosystem II particles in A. nidulans. This protein might have a dual role: acting as an electron transferring protein in photosynthesis in the presence of certain metal ions (M3+ > M2+ > M+) and as an l‐amino‐acid oxidase in the absence of these.
Preparation of Apoprotein from Spinach Ferredoxin‐NADP+ Reductase
Authors: ZANETTI Giuliana , CIDARIA Dante , CURTI Bruno 
Published: March 2005

Ferredoxin‐NADP+ reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl2 at pH 7.5 and 2°C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCl. The apoprotein is stable for at least a week at −20°C.
The release of the prosthetic group from the protein by the Ca2+ ions is a multi‐step process. Three different effects of these ions are identifiable: (a) a rapid 20–25% inhibition of catalytic activity, probably caused by an increase in the ionic strength of the medium; other cations can produce it as well; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the FAD is finally released from the protein; (c) complete conversion of FAD to FMN, which blocks reconstitution to holoenzyme, caused by the well‐known hydrolytic action of Ca2+ ions on the pyrophosphate bridge of FAD
Binding of FAD by the apoferredoxin‐NADP+ reductase is very rapid and it is complete in a few minutes even at 0°C. A Kd of 3.4 × 10−9 M has been determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.
Chemical Modification Studies on Ricinus communis (Castor Bean) Agglutinin
Authors: KHAN M. Islam , SUROLIA Avadhesha 
Published: March 2005

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide‐binding properties was studied.Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide‐binding activity of Ricinus agglutinin.Modification of tryptophyl, arginyl and carboxyl‐group‐containing residues did not lead to any loss in the activity of the agglutinin.Acetylation of tyrosyl groups with N‐acetylimidazole strongly reduced the hemagglutinating and saccharide‐binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin.The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.
The Structural Isomerisation of Human‐Muscle Adenylate Kinase as Studied by 1H‐Nuclear Magnetic Resonance
Authors: KALBITZER Hans Robert , MARQUETANT Rainer , RÖSCH Paul , SCHIRMER R. Heiner 
Published: March 2005

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H‐nuclear magnetic resonance spectroscopy. The C‐2 and C‐4 proton resonances of the active‐center histidine His‐36 could be identified; the pK of His‐36 was determined as 6.1. The pK of His‐189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947–6954].A pH‐dependent structural isomerization as shown by X‐ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37–45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5′)pentaphospho(5′)adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s−1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion‐controlled process, Kon being 108 M−1 s−1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X‐ray diffraction in parallel, is suitable for analysing the induced fit postulated by Jencks for all kinase‐catalyzed reactions.