Dang Ba Pho

Octopine déshydrogénase. Purification et propriétés catalytiques.

Abstract Octopine dehydrogenase: Purification and catalytic properties 1. I. Octopine dehydrogenase, an NAD+ enzyme, which catalyzes the dehydrogenation of octopine into arginine plus pyruvale, has been purified from muscles of Pecten maximus. It is homogeneous on analytical ultracentrifugation and disc electrophoresis. 2. 2. The enzyme contains no such metal cofactor as Fe2+, Mn2+, Zn2+. 3. 3. The Michaelis constants are: 1.5·10−3 M for octopine, arginine and pyruvate, 1.5·10−4 M for NAD+ and 4·10−5 M for NADH. 4. 4. Substrate analogs with a guanidyl (guanidinobutane) or a carboxyl group (valeric acid) or both of them (δ-guanidinovaleric acid) are competitive inhibitors of the octopine forming reaction. In the dehydrogenation reaction, compounds with only one of these groups are competitive inhibitors whereas those possessing both guanidyl and carboxyl groups are not.

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.

Spectrophotometric studies of binary and ternary complexes of octopine dehydrogenase.

Abstract 1. 1. The interaction of octopine dehydrogenase of Pecten maximus with coenzymes and substrate analogues was studied by a spectrophotometric difference method. 2. 2. The coupling of the enzyme with coenzymes gives rise to spectral changes in the ultraviolet region similar to those given by a protonation of the adenine ring; in the visible region the difference spectrum presents a red shift for the reduced nicotinamide. 3. 3. The binding of substrate analogues to the binary complexes produces a specific shift in the 288–300-nm region which should correspond to a red shift of the tryptophan residue spectrum. This specific shift is produced only by the substrate analogues that possess both the carboxylic and the guanidino groups. 4. 4. The lack of interaction of arginine with apoenzyme is a possible direct evidence of an obligatory, ordered reaction sequence in enzymic reactions.

Arginine oxygenase decarboxylante: V. Purification et nature flavinique

1. 1. Arginine oxygenase (decarboxylating) from Streptomyces griseus, the enzyme which converts l-arginine into γ-guanidobutyramide, has been purified. 2. 2. The enzyme contains a flavin cofactor identified as FAD. 3. 3. Analytical ultracentrifugation seems to demonstrate reversible dissociation of the protein.

Cytoskeletons of ADP- and thrombin-stimulated blood platelets: Presence of a caldesmon-like protein, α-actinin and gelsolin at different steps of the stimulation

Comparative analyses of the cytoskeletons of resting and stimulated platelets point out the involvement of a 79 kDa polypeptide in the activation step and its increased incorporation during aggregation. It appears as a doublet and cross‐reacts with an antibody to chicken gizzard caldesmon, whereas no 150 kDa immunoreactive form was detected. α‐Actinin and gelsolin were detected only in the aggregation step.

Evidence for the presence of tropomyosin in the cytoskeletons of ADP‐ and thrombin‐stimulated blood platelets

Stimulation of porcine platelets with ADP or thrombin and subsequent analyses of their cytoskeletons by SDS‐polyacrylamide gel electrophoresis have shown the presence of a 30.5‐kDa polypeptide in the cytoskeletons of activated as well as aggregated platelets. This polypeptide comigrates with pure porcine platelet tropomyosin in SDS gels, their mobilities being similarly and markedly decreased in the presence of 6 M urea. One‐dimensional peptide mapping after limited proteolysis by Staphylococcus aureus protease gives the same pattern for pure tropomyosin and the 30.5‐kDa polypeptide. This latter may thus be identified as the porcine platelet tropomyosin subunit, the role of which may not be solely structural.

L-Arginine oxygenase decarboxylante IV. Incorporation de 18O dans la γ-guanidinobutyramide

Summary 1. Arginine oxygenase (decarboxylating) (EC Class 1.13) isolated from Streptomyces griseus has been incubated with L -arginine in the presence of either 18 O 2 and H 2 16 O or 16 O 2 and H 2 18 O. 2. The reaction product, γ -guanidinobutyramide, has been purified using Amberlite IRC-50, isolated and crystallized in form of the chloroplatinate. 3. Isotopic analysis has clearly shown that the 18 O incorporated into the reaction product is supplied by 18 O 2 and not by H 2 18 O. According to its mode of reaction, the enzyme of S. griseus is to be grouped with the oxygenases. From its specificity, it can be identified as L -arginine oxygenase (decarboxylating).

Essential carboxyl groups in yeast hexokinase

Although a great deal of kinetic data are available on yeast hexokinase and several schemes have been proposed, the mechanism of its action is far from being understood [ 1,2] . It is generally agreed, however, that the transphosphorylation step requires the formation of a ternary complex between the enzyme and its substrates, ATP-Mg and D-glucose. Although the pH dependence of the enzyme activity suggests that at least one ionisable group, such as the imidazole of histidine or the y-carboxyl group of aspartic or glutamic acids, takes part in the catalytic process [3, 41, the nature of the amino acid residues involved in enzymatic catalysis or substrate binding remains to be determined. According to the results of Grouselle et al. [5], as well as our own unpublished results, the histidine residues do not appear to be associated either with the catalytic step or with the enzyme-substrate interactions. Thus, the inactivation observed when the enzyme is carboethoxylated is caused by a conformational change in the enzyme protein rather than by the modification of an essential histidine residue. It seemed, therefore, important to investigate the effect of specific carboxyl group reagents upon the enzyme activity. Water soluble carbodiimides have been shown to be good reagents for this purpose [6]. This paper reports some preliminary results obtained by chemical modification of essential carboxyl groups in yeast hexokinase.

Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.

Sites actifs de la l-arginine decarboxy-oxydase

1. 1. l-Arginine decarboxy-oxidase (l-arginine oxygenase (decarboxylating)) is strongly inhibited by the more alkaline derivatives (agmatine, guanidobutane, galegine), less strongly by δ-guanidovaleric and γ-guanidobutyric acids, and only slightly by γ-guanidobutyramide and d-arginine. These inhibitors are competitive with the substrate, l-arginine. In the same order these derivatives prevent the enzyme from inactivation by O2. 2. 2. l-Arginine decarboxy-oxidase is strongly inhibited by p-chloromercuribenzoate. This inhibition is antagonized neither by the more alkaline guanidine derivatives nor by d-arginine. The competition is effective with reduced glutathione, with guanidoacids and with α-amino acids. 3. 3. The enzyme is likely to possess two active sites. One of them is auto-oxydable and binds with the guanidine group; the other, which reacts with p-chloromercuribenzoate, seems to bind with the α-NH2 and CO2H groups.