Anna Olomucki

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.

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.

Isolation and physico-chemical properties of blood platelet α-actinin

Abstract A procedure for the isolation of α-actinin from human blood platelets is described. Typical yields were 10–13 mg from 48 g of frozen platelets. The purified platelet α-actinin has many physico-chemical properties (molecular weight in native state, molecular weight in denaturing conditions, Stokes radius, ellipticities at 208 and 221 nm) similar to those of muscle α-actinins. However, in contrast to muscle α-actinins, it is composed of isoforms containing subunits of slightly different molecular weights and its effect on actin gelation is calcium-sensitive. These two characteristics are common to other known non-muscle α-actinins.

Arginine décarboxy-oxydase: I. Caractères et nature de l'enzyme

Abstract 1. 1. The purification and properties of arginine decarboxy-oxidase present in Streptomyces griseus (Waksman) are described. The enzyme is highly specific. 2. 2. The stoichiometry of the enzymic reaction has been investigated. Thus, the ratio of arginine oxidized to oxygen absorbed to carbon dioxide evolved is 1:1:1. This ratio is not modified by the presence of catalase. The reaction does not occur anaerobically in the presence of ferricyanide. 3. 3. The enzyme is inactivated by oxygen. In the presence of arginine as substrate this inactivation is reduced. Sulfhydryl reagents similarly show less inhibitory activities in the presence of the substrate. 4. 4. Besides Str. griseus, several other species of Acetomyces have been shown to have arginine decarboxy-oxidase activity.

Induction et spécificité des enzymes de la nouvelle voie catabolique de l'arginine

Resume 1. Arginine decarboxyoxidase (arginine oxygenase (decarboxylating)), guanidinobutyramidase (guanidinobutyramide amidohydrolase) and guanidinobutyrate ureohydrolase from Streptomyces griseus , which catalyze the first three stages of the newly demonstrated catabolic pathway leading from arginine to succinic semialdehyde, were shown to be induced by arginine added to the culture medium. The degree of induction is not the same for all three enzymes. 2. 2. Under the same conditions, arginase is not induced. Owing to the absence of this enzyme and of l -amino acid oxidase, the catabolic pathway initiated by the oxidation of arginine to γ-guanidinobutyramide is the only way of utilizing of arginine in S. griseus . 3. 3. Guanidinobutyrate ureohydrolase is a strictly specific enzyme; it does not act upon d - and l -arginine nor upon various guanidine derivatives.

The effect of serum vitamin D-binding protein on polymerization and depolymerization of actin is similar to the effect of profilin on actin.

The mechanism of the interaction between two genetically determined serum vitamin D-binding protein forms and the muscle skeletal actin was investigated. Vitamin D-binding protein was isolated in a good yield from human serum, using immunoaffinity chromatography. 16 mg of pure vitamin D-binding protein were obtained from 100 ml of serum. The interaction between purified vitamin D-binding protein and skeletal muscle actin was studied by viscosity, delta A (232 nm) measurements and by electron microscopy. The effect of vitamin D-binding protein on actin polymerization is characterized by the decrease of the nucleation and elongation rates and by the decrease of the final concentration of polymerized actin in the steady state. The depolymerizing effect is not the result of direct action on vitamin D-binding protein on F-actin but rather of an increased concentration of the complex of the former protein with G-actin. The characteristics of the vitamin D-binding protein and profilin interactions with actin are similar. Both proteins seem to react only with G-actin.

Isolation and characterization of human blood platelet gelsolin.

A 90‐kDa protein‐actin stable complex was purified from blood platelets by a short and efficient procedure giving at the same time actin used in polymerization assays. 90‐kDa protein free of actin was prepared from the complex by 8 M ureau treatment and renaturation. By its molecular mass, immunological cross‐reactivity with macrophage gelsolin and its effect on G‐ and F‐actin the 90‐kDa protein appears as the platelet gelsolin.

Susceptibility of platelet α-actinin to a Ca2+-activated neutral protease

Purified α-actinin from human platelets was digested with Ca2+-activated protease from muscle. The a subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, α-actinin is cleaved by the endogenous Ca2+-activated protease.

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.