[The role of terminal DNA groups in the activation of (ADP-ribose)polymerase]. / Rol' kontsevykh grupp DNK v aktivatsii (ADP-riboza)polimerazy.

Some peculiarities of activation of (ADP-ribose) polymerase by DNA fragments were studied. DNA fragments were produced by the digestion of calf thymus DNA by micrococcal nuclease and with a subsequent enzymatic modification of their end groups by nuclease S1, polynucleotide kinase of phage T4 and alkaline phosphatase. The dependence of the activating effect of DNA on the chemical structure of its end groups was established. It was shown that the terminal phosphate groups are involved in the formation of a catalytically active complex of (ADP-ribose) polymerase with DNA.

[Activation of (ADP-ribose) polymerase by DNA fragmented by micrococcal nuclease and DNAase I]. / Aktivatsiia (ADP-riboza) polimerazy DNK, fragmentirovannoi mikrokokkovoi nukleazoi i DNKazoi I.

Activation of (ADP-ribose) polymerase by DNA fragments obtained by digestion of calf thymus DNA with micrococcal nuclease and DNAase I was studied. It was found that activation of the enzyme is due to its interaction with the terminal parts of double-stranded DNA fragments, the level of activation being independent of the size of DNA fragments.

[Structural organization of a hexamer of glutamate dehydrogenase. 3. Effect of the coenzyme and substrate on the urea-induced dissociation and inactivation of the hexamer]. / Strukturnaia organizatsiia geksamera glutamatdegidrogenazy. 3. Vliianie kofermenta i substrata na indutsiruemye mochevinoi dissotsiatsiiu i inaktivatsiiu geksamera.

Effects of coenzyme (NADH) and substrate (2-oxoglutarate) on the urea-induced dissociation and inactivation of immobilized phosphopyridoxyl derivative of bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) have been studied. Urea at concentration 3.0 to 4.0 M in the presence of NADH induced dissociation of the enzyme's hexamer to catalytically inactive immobilized dimer. In the presence of both NADH and 2-oxoglutarate at the urea concentration 1.0 to 2.0 M the hexamer dissociated to the conformationally stable immobilized trimer possessing 60% catalytic activity of the hexamer. Studies of regulatory properties of the immobilized trimer showed that the allosteric inhibition of glutamate dehydrogenase by GTP was realized on the level of trimers, where the subunits interact through identical heterological contacts.

[Structural organization of glutamate dehydrogenase hexamer. 1. Immobilization on sepharose as a model for analysis of structural-functional characteristics of the enzyme]. / Strukturnaia organizatsiia geksamera glutamatdegidrogenazy. 1. Immobilizatsiia na sefaroze s tsel'iu polucheniia modeli dlia izucheniia strukturno-funktsional'nykh osobennostei fermenta.

Bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) and its radioactive phosphopyridoxyl derivative were covalently immobilized on Sepharose CL-4B with different degrees of cyanogen bromide activation. The catalytical and regulatory properties of the immobilized samples of the enzymes were studied. It was shown that the enzymes were immobilized through a single subunit of hexamer when sepharose was activated by small amounts of cyanogen bromide (less than 5 mg per 1 ml of gel). In this case, the immobilization did not alter the catalytical and regulatory properties of glutamate dehydrogenase. The immobilized radioactive phosphopyridoxyl derivative of glutamate dehydrogenase completely imitated the immobilized native enzyme and can be used as a convenient model for structural and functional investigation of catalytically active hexamer of glutamate dehydrogenase.

[Structural organization of glutamate dehydrogenase. 2. Inactivation and dissociation of immobilized hexamer induced by urea]. / Strukturnaia organizatsiia geksamera glutamatdegidrogenazy. 2. Izuchenie inaktivatsii i dissotsiatsii immobilizovannogo geksamera pod vliianiem mocheviny.

The urea-induced inactivation and dissociation of catalytically active hexamer of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) from bovine liver were studied using radioactive phosphopyridoxyl derivative of the enzyme immobilized on cyanogen bromide-activated Sepharose CL-4B. It is shown that at neutral pH (7.0-7.8) urea causes dissociation of glutamate dehydrogenase to directly yield catalytically inactive immobilized monomers rather than hexamer's stable fragments at the same time. At pH 8.9 or 5.6 the urea-induced is accompanied by the formation of conformationally stable immobilized dimers or trimers, respectively. The trimers are catalytically active, whereas the dimers did not exhibit any enzymatic activity. The data obtained led to suggestion that the hexamer consists of three either equivalent dimers (3 alpha 2) or of two equivalent trimers (2 alpha 3).

[Cooperative inactivation of glutamate dehydrogenase of 2,2,6,6- tetramethyl-4-oxopiperidine-1-oxyl. Interpretation of results within the scope of a hexamer model with equivalent subunit orientation]. / Kooperativnaia inaktivatsiia glutamatdegidrogenazy 2,2,6,6- tetrametil-4-oksopiperidin-1-oksilom. Interpretatsiia resul'tatov v ramkakh modeli geksamera s ékvivalentnoi orientatsiei sub''edinits.

It was shown that the blockage of epsilon-amino group of Lis-126 residue by 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TMPO) leads to the cooperative inactivation of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3). The data concerning cooperative inactivation of the enzyme are interpreted by the model of hexamer with identical orientation of subunits. It was shown that the modification of any of enzyme subunits is accompanied by an inactivation of the hexamer's fragment which is a dimer, with subunits interacting reciprocally by means of isological contacts.

[Reaction of poly(ADP)-ribosylation of histone H1 in the presence of P1,P4-bis(5'-adenosyl)tetraphosphate and its phosphonate analogs]. / Izuchenie reaktsii poli(ADP)-ribozilirovaniia gistona H1 v prisutstvii P1,P4-bis(5'-adenozil)tetrafosfata i ego fosfonatnykh analogov.

Effects of P1,P4-bis(5'-adenosyl)tetraphosphate and its phosphonate analogs on the ADP-ribosylation of H1 catalyzed by bovine testis ADP-ribose polymerase was investigated. Analogs App[CH(COCH3)]ppA and Ap[CH2]pppA as well as Ap4A inhibited poly(ADP)-ribosylation of histone H1 and at the same time accepted the ADP-ribosyl moiety of NAD. It was shown that inhibition of ADP-ribosylation of histone H1 is due to the competition of nucleotides with histone H1 for accepting ADP-ribosyl moiety of NAD on the one hand, and alteration of acceptor properties of the histone H1 on the other.

[Isolation of (ADP-ribose)polymerases from the calf testis and thymus and comparison of their basic physico-chemical properties]. / Vydelenie (ADP-riboza)polimeraz iz semennikov i timusa byka i sravnenie ikh osnovnykh fiziko-khimicheskikh svoistv.

Highly purified preparations of (ADP-ribose) polymerase were obtained from calf testis and thymus by chromatography on DNA-cellulose, hydroxyapatite and gel filtration. It was shown that the enzymes isolated from both sources under identical conditions have similar values of Mr, Vmax and Km in the reactions of autoribosylation and poly(ADP-ribosylation) of histone H1 as well as similar pH-dependencies of the catalyzed reactions.

[Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the cooperative type of inhibition by GTP]. / Modifikatsiia glutamatdegidrogenazy piridoksal'-5'-fosfatom. Izuchenie kooperativnogo kharaktera ingibirovaniia GTP.

The character of allosteric inhibition of glutamate dehydrogenase by GTP was studied. The derivative of the enzyme not capable of being polymerized was taken as a model. It was shown that: in the absence of NADH every protomer of this derivative can bind one molecule of GTP; in the presence of NADH the additional binding site for GTP was induced; the modification of the enzyme derivative by pyridoxal-5-phosphate in the presence of NADH and alpha-ketoglutarate blocked the NADH-induced GTP binding site and the disappearance of positive kinetic cooperativity induced by GTP was observed; to achieve the inhibitory action of GTP the binding of the effector to only one (NADH-induced) site was enough; the role of GTP binding to the NADH-induced site is to provide better affinity of the effector to the "inhibitory" centre; the positive kinetic cooperativity of inhibition of glutamate dehydrogenase by GTP depends probable on the cooperative character of interaction between the two molecules of GTP to each protomer of the enzyme.

[Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the structural organisation of the hexamer and its possible role in the realization of GTP action]. / Modifikatsiia glutamatdegidrogenazy piridoksal'-5'-fosfatom. Izuchenie strukturnoi organizatsii geksamera i ego vozmozhnoi roli v realizatsii deistviia GTP.

The catalytic and regulator properties of glutamate dehydrogenase by modification of Lys-126 residue by puridoxal-5'-phosphate was studied. The phosphopyridoxyl derivative of the enzyme with blocked NADH-induced binding site of GTP not capable of being polymerized was taken as a model. It was shown that: blocking the epsilon-amino group of Lys-126 residue brings to a simultaneous inactivation of the enzyme and desensibilization of its residual activity to GTP action; the modification of Lys-126 residue and resulting inactivation of the enzyme and desensibilization to GTP action were non-cooperative processes, with equal values of pseudofirst order rate constants; modification of Lys-126 residue of any of hexamer's protomer results in the desensibilization to GTP action on one of the contacting, catalytically active protomers. The experimental dependence of the inhibition degree of the enzyme by GTP upon the average number of modified residues of Lys-126 is explained by the model of the hexamer of glutamate dehydrogenase with identical interlocation of any of the protomers in relation to the one in contact.

[Purification of glutamate dehydrogenase from the rabbit liver and study of its major physico-chemical properties]. / Ochistka glutamatdegidrogenazy pecheni krolika i izuchenie osnovnykh fiziko-khimicheskikh svoistv.

Highly purified preparations of glutamate dehydrogenase were obtained from mitochondrial and cytoplasmic fractions of rabbit liver by affinity chromatography on CL-Sepharose 4B modified by adenosine diphosphate. Some physico-chemical properties of the purified enzymes (e. g., specific activity, molecular weight, quaternary structure, stability against denaturating effect of urea, pH optimum of catalyzed reactions, Km values for substrates and coenzymes) were found to be identical. The sole difference was detected in the ability of enzyme preparations to be activated by adenosine diphosphate. The activation of the cytoplasmic enzyme is 160%, that of mitochondrial glutamate dehydrogenase is 230-240% under the same conditions.

[Identification of a glutamate dehydrogenase amino acid residue modified by 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl. Study of the type of inactivation of a catalytically active enzyme oligomer in modification]. / Identifikatsiia aminokislotnogo ostatka glutamatdegidrogenazy, modifitsiruemogo 2,2,6,6-tetrametil-4-oksopiperidin-1-oksilom. Izuchenie kharaktera inaktivatsii kataliticheski aktivnogo oligomera fermenta pri modifikatsii.

It has been shown that 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl selectively blocks epsilon-amino group of Lys126 residue in bovine liver glutamate dehydrogenase (L-glutamate NAD(P) oxydoreductase, EC 1.4.1.3). Modification of this residue in one of the six promoters of catalytically active hexamer is accompanied by the loss of about half of the enzymatic activity. The enzyme inactivation caused by modification has a cooperative character.

[Effect of bivalent copper ions on the catalytic properties of bovine liver glutamate dehydrogenase]. / Vliianie dvukhvalentnogo iona medi na katalitichskie svoistva glutamatdegidrogenazy pecheni byka.

The effects of bivalent copper ions on the activity of bovine liver glutamate dehydrogenase (L-glutamate NAD (P) oxidoreductase, EC 1.4.1.3) were studied. Cu2+ effectively interacts with the enzyme; this interaction is accompanied by a loss of the enzyme activity in the reaction of reductive amination of alpha-ketoglutarate. The data obtained are indicative of a similar type of the enzyme inhibition by GTP and copper ions.

[Bovine liver glutamate dehydrogenase modified by 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl: enzymatic activity and catalytic properties]. / Modifikatsiia glutamatdegidrogenary pecheni byka 2,2,6-6-tetrametil-4-okso-piperidin-1-oksilom. Vliianie na aktivnost' i kataliticheskie svoistva.

When modified by 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TMPO) bovine liver glutamate dehydrogenase (L-glutamate NAD(P) oxidoreductase, E. C. 1.4.1.3) looses its catalytical activity and sensitivity to allosteric inhibitor GTP. The stoicheiometry of the binding of TMPO to glutamate dehydrogenase has been studied--each protomer bound one molecule of TMPO. It is supposed that TMPO reacts with lysine residue located in the enzyme's active center.

[Conformational stability of glutamate dehydrogenase and its complexes with specific ligands]. / Konformatsionnaia stabil'nost' glutamatdegidrogenazy i ee kompleksov so spetsificheskimi ligandami.

The isotermic denaturation of glutamatdehydrogenase (GDH) and its complexes with co-enzymes, substrates and allosteric regulators under the action of urea was studied. It was shown that the reaction of the enzyme with an allosteric inhibitor GTP is accompanied by a decrease in conformational stability of the catalytically active hexsamer GDH. Formation of a complex with the allosteric activator ADP increases the conformational stability of the enzyme. Studies on the isotermic unfolding of GDH in the presence of various phosphoric ethers of adenosine gave evidence that the stabilizing effect of ADP is based on the reaction of the enzyme with the adenine base of the regulator.

[Purification and some physico-chemical properties of glutamate dehydrogenase from beef brain]. / Ochistka glutamatdegidrogenazy iz mozga byka i ee nekotorye fiziko-khimicheskie svoistva.

A procedure for purification of glutamate dehydrogenase (GDH; L-glutamate NAD(P) oxidoreductase, EC 1.4.1.3) from beef brain has been developed. The enzyme preparation obtained has the specific activity of 6.7 units per mg of protein (192-fold enhance with a 30% yield of total activity of the homogenate). In some of its physico-chemical properties (pH optimum of catalyzed reactions, molecular weight, subunit structure, thermal stability) the brain GDH is identical to the enzyme from beef liver. The Km values for most of the coenzymes and substrates for the enzyme studied do not exceed those for beef liver enzyme more than 1,5--2-fold. The only exception is the Km value for glutamate, which in the case of brain GDH is 4 times less than that for the liver enzyme. The results obtained suggest that upon interaction with NAD the brain GDH reveals a relatively higher affinity for L-glutamate and L-ketoglutarate as compared to the liver enzyme.

[The circular dichroism spectrum of dinucleoside phosphate analogs]. / Spektry krugovego dikhroizma analogov dinukleozidfosfatov

Circular dichroism spectra of 11 analogues of the dinucleoside phosphate containing achiral 3'-terminal monomers have been measured at several pH values, various temperatures and various concentrations of ethanol. The conformation of analogues studied has been shown to by very similar to that of natural compounds. Comparison of the results obtained with the circular dichroism spectra of the corresponding natural compounds indicates that Cotton effect arises from monomeric circular dichroism, at least in main features. The exciton interaction is relatively small.