[The structural membrane control of the reduction of exogenous quinones catalysed by mitochondrial 2-methyl-1,4-naphthoquinone reductase]. / Strukturno-membrannyi kontrol' vosstanovleniia ekzogennykh khinonov, kataliziruemogo mitokhondrial'noi 2-metil-1,4-naftokhinon-reduktazoi.

The catalytic parameters of mitochondrial 2-methyl-1, 4-naphthoquinone-reductase were studied. Maximal velocity and Michaelis constant of quinone-reducing reaction were shown to depend strongly on the functional state of mitochondria (I, II and IV Chance's states). The changes in catalytic parameters were found for four quinone substrates essentially varying in their structures. For the same conditions the Michaelis constant of isolated enzyme remains unchanged. The data obtained indicate the possibility to control the activity and substrate specificity by structural rearrangements within the biological membrane.

[Oxidation of 4-anilino-5-methoxydioxybenzene-1,2 in complex biochemical systems possessing superoxide dismutase activity]. / Okislenie 4-anilino-5-metoksi-1,2-dioksibenzola v slozhnykh biokhimicheskikh sistemakh, obladaiushchikh superoksiddismutaznoi aktivnost'iu.

The kinetics of 4-anilino-5-methoxydioxybenzene-1,2 (AMOBQH2) autoxidation in biochemical systems possessing the superoxide dismutase activity were studied. The autoxidation of AMOBQH2 is affected by superoxide dismutase, which is indicative of participation of the superoxide radical in this process. The main kinetic effect of superoxide dismutase consists in a decrease of the effective rate constant for AMOBQH2 autoxidation. Peroxidase releases the superoxide dismutase inhibition of AMOBQH2 autoxidation. The data obtained are discussed in terms of a biochemical mechanism of action of biologically active aminoaromatic derivatives of o-benzoquinone.

[Oxidation of some substrates in the presence of NAD(P)-dependent oxidoreductases and 4-anilino-5-methoxybenzoquinone-1,2 with transfer of reducing equivalents to oxygen]. / Okislenie nekotorykh substratov pri uchastii NAD(P)-zavisimykh oksidoreduktaz n 4-anilino-5-metoksi-1,2-benzokhinona s perenosom vosstanovitel'nykh ekvivalentov na kislorod.

A reaction sequence for oxidation of L-lactate, ethanol, D-glucose-6-phosphate, and isocytrate by oxygen in the presence of NAD(P)-dependent oxidoreductases and 4-anilino-5-methoxybenzoquinone-1,2 was studied. A complete reaction sequence includes (1) oxidoreduction of a corresponding substrate with a specific NAD(P)-dependent dehydrogenase and formation of NAD(P)H (2) reduction of AMOBQ, AMOBQHa formation in the oxidoreduction of NAD(P)H and AMOBQ, catalized by menadione reructase (EC 1.6.99.2); (3) oxygen reduction with AMOBQH2 in a spontaneous or enzymic reaction. AMOBQ plays a catalytic role in the transfer of reducing equivalents to oxygen in the above--menshioned systems. Possible biochemical mechanism for aminoaromatic ortho-benzoquinone derivatives is discussed.

[Affinity chromatography of menadione reductase on Sepharose with aminoaromatic and aminoaliphatic quinone ligands]. / Affinnaia khromatografiia menadionreduktazy na sefaroze s aminoaromaticheskimi i aminoalifaticheskime khinonnymi ligandami.

New modifications of Sepharoses with the ligands--4-hexamethylenimino-5-methoxy-1.2-benzoquinone (I), 4-(p-imminomethylaniline)-5-methoxy-1.2-benzoquinone (II) and 4-(p-iminomethylaniline)-5-methoxy-1.2-dihydroxybenzene (III) are described and their absorption properties with respect to menadione reductase (EC 1.6.99.2) investigated. The modification with ligand (II) affinitely adsorbs menadione reductase; the enzyme remains adsorbed in the presence of high concentration of electrolytes and within the pH range of 6.0-7.5. The elution is specific and is achieved by NAD (P) H but not by NAD (P). After chromatography the enzyme is homogeneous as shown by disc-electrophoresis in polyacrylamide gel. Under similar conditions Sepharoses with ligands (I) and (III) but poorly bind the enzyme. It is assumed that an essential role in the adsorption mechanism belongs to dispersion forces and to formation of a molecular charge-transfer complex between the aminoaromatic quinone ligand and menadione reductase active center.

[Affinity chromatography of menadione reductase]. / Affinnaia khromatografiia menadionreduktazy.

The preparation of affinity adsorbents for menadione reductase (EC 1.6.99.2), using CNBr-activated Sepharose 4B and activated CH-Sepharose 4B, is described. The adsorbents used are the sepharoses modified by 4-anilino-5-methoxybenzoquinone-1,2. Bound menadione reductase is specifically eluted with NAD(P)H, while NAD(P) has no effect on the enzyme. The enzyme yield of 83--95% and a 102-fold purification are achieved. Disc-electrophoresis reveals one protein fraction identified with menadione reductase and a separate inactive fraction. It is assumed that the specific binding of menadione reductase to the adsorbent is due to the formation of a complex between the oxidized enzyme and the quinone. This complex breaks down in the presence of NAD(P)H.

[Comparative analysis of cytosolic and solubilized mitochondrial menadione reductases from rat liver]. / Sravnitel'noe izuchenie tsitozol'noi i soliubilizirovannoi mitokhondrial'noi menadionreduktaz iz pecheni krysy.

The kinetic properties of cytosolic and solubilized mitochondrial menadione reductases (EC 1.6.99.2) from rat liver were compared. The mechanism of the reaction of cytosolic and mitochondrial menadione reductases with NADH and 4-anilino-5-methoxy-1,2-benzoquinone (AMOBQ) as substrates obeys the "ping-pong" kinetics. AMOBQ is a competitive inhibitor of cytosolic menadione reductase (Ki = 219 microM). Both menadione reductases have similar or identical values of true and effective kinetic constants and similar electrophoretic mobilities.

[Isolation, purification and structure of molecules of a vasoactive component from rabbit skeletal muscle]. / Vydelenie, ochistka i struktura molekuly vazoaktivnogo komponenta iz skeletnykh myshts krolika.

A low molecular weight factor with pronounced vasoactive properties has been isolated from rabbit muscle. The procedure for lyophilized tissue extract purification included fractional methanol extraction with subsequent column chromatography on TSK-GEL Toyopearl, DEAE-Toyopearl 650 ion-exchanger and Sephadex G-10. The resulting preparation was homogeneous as evidenced from reversed phase HPLC. The structure of the factor was examined by using UV- and IR-spectrophotometry as well as by PMR and 13C-NMR. It was found that the vasoactive factor includes an inosine nucleotide structure covalently bound to a di- or tri-alanine peptide while the phosphate group is free. It is suggested that the binding of the peptide to the inosine moiety occurs via a C-terminal carboxyl of the peptide and pentose hydroxyls. It seems probable that the vasoactive effect is a result of esterification of the purine nucleotides with the peptide.

[Properties and reaction mechanism of mitochondrial menadione reductase]. / Svoistva i kineticheskii mekhanizm deistviia mitokhondrial'noi menadionreduktazy

An isolation procedure of mitochondrial menadione reductase from rat liver using an ethanol-ether extraction for solubilization of the enzyme is described. The enzyme was purified 930-fold. The molecular weight of mitochondrial menadione reductase is 62,000. According to spectroscopic and enzymic analysis the prosthetic group of the enzyme was identified as FAD. Mitochondrial menadione reductase is inhibitied by dicumarol and p-chloromecuribenzoate. The enzyme is characterized by a group substrate specificity towards quinones. A high catalytic activity of menadione reductase towards 4-aniline-5-methoxy-1,2-benzoquinone (AMOBQ), and 4-N-(p-sulfoanilino)-5-methoxy-1,2-benzoquinone (AMOBQS) as acceptors was demonstrated. It was shown that the reduction of these orto-benzoquinones by NAD(P) H follows the "ping-pong" kinetics. The kinetic constants for NAD(P)H,AMOBQ and and AMOBQS were determined.

[The use of certain new O-benzoquinone derivatives as acceptor substrates in enzymatic oxidation of NADH]. / Ispol'zovanie Novykh Proizvodnykh O-benzokhinona v kachestve aktseptiruiushchikh substratov pri fermentativnom okislenii NADH

A possibility of the use of new aliphatic and aminoarylic o-benzoquinone derivatives as acceptor substrates in NADH oxidation catalyzed by NAD(P)H: (acceptor)-oxidoreductase (EC 1.6.99.2) was demonstrated. The kinetic mechanism of this reaction was studied in a monosubstrate approach and the effective kinetic constants were determined. The stability of these o-benzoquinone derivatives in the oxidation-reduction reactions, including anzymic reduction and subsequent auto-oxidation of their diphenolic forms by oxygen, was substantiated using spectrophotometric techniques.