[Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Condtions].

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

[Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s].

The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis.

[Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

[Structural-functional transformation of the malate dehydrogenase system of the bacterium Sphaerotilus sp. strain D-507 depending on nutritional mode].

High-purity preparations of malate dehydrogenase (EC 1.1.1.37) were obtained by multistage purification from the bacterium Sphaerotilus sp. strain D-507 growing under different conditions. Under organotrophic conditions, the enzyme was dimeric; under mixotrophic conditions, dimeric and trimeric. On the basis of studied properties of the enzyme preparations, data on the activity of enzymes of the glyoxylate and tricarboxylic-acid cycles, and analysis of published data, it can be concluded that malate dehydrogenase isoforms are implicated in the adaptive response of bacteria to changing culturing conditions.

[Regulation of carbon flows in the tricarboxylic acid cycle-glyoxylate bypass system in Rhodopseudomonas palustris under different growth conditions].

The functional roles of the malate dehydrogenase (MDH) tetrameric and dimeric isoforms in the metabolism of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris, strain f-8pt, was studied with the use of specific inhibitors. It was shown that the enzyme tetrameric form allows the functioning of the glyoxylate cycle and the dimeric form provides for the operation of the tricarboxylic acid cycle.

[Isolation and properties of malate dehydrogenase from meso- and thermophilic bacteria].

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 +/- 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 +/- 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.

Physicochemical properties of malate dehydrogenase from the bacterium Rhodopseudomonas palustris strain f8pt.

Electrophoretically homogenous isoforms of malate dehydrogenase with different quaternary structure were prepared from Rhodopseudomonas palustris strain f8pt cultured photolithoheterotrophically on malate and acetate. By selective inhibition of the tricarboxylic acid cycle or glyoxylate cycle, it was shown that the dimeric isoform of the enzyme is responsible for Krebs cycle functioning and the tetrameric isoform is involved in functioning of the glyoxylate cycle.

Malate dehydrogenase from the thermophilic bacterium Vulcanithermus medioatlanticus.

Thermostable dimeric malate dehydrogenase (MDH) was isolated from the microorganism of hydrothermal vents Vulcanithermus medioatlanticus. The enzyme was electrophoretically homogeneous and possessed the specific activity of 6.9 U/mg. The large molecular weight of the subunits (55 kD) is likely to provide the rigidity of the enzyme structure (the activation energy of the enzymatic reaction is 32.6 kJ/mol). The thermophilic MDH differs little from the mesophilic enzyme in terms of kinetic and regulatory characteristics.

[Role of malate dehydrogenase isoforms in the regulation of anabolic and catabolic processes in the colorless sulfur bacterium Beggiatoa leptomitiformis D-402]. / Rol' izoform malatdegidrogenazy v reguliatsii anabolicheskikh i katabolicheskikh protsessov u bestsvetnykh serobakterii Beggiatoa leptomitiformis D-402.

The functional role of tetrameric and dimeric isoforms of malate dehydrogenase in the carbon metabolism of the colorless sulfur bacterium Beggiatoa leptomitiformis, strain D-402, was studied. This strain can grow both lithotrophically and organotrophically. By using the inhibition analysis, the tetrameric isoenzyme was shown to operate in the glyoxylate cycle and the dimeric one was found to be involved in the TCA cycle. The dynamics of the dimeric isoenzyme conversion to the tetrameric isoform was found to be determined by the rate of thiosulfate oxidation. The regulation of the carbon metabolism in Beggiatoa leptomitiformis is supposed to be accomplished by means of structural and functional changes in the protein molecule of malate dehydrogenase.

[Isolation, purification, and properties of malate dehydrogenases from sulfur bacteria Beggiatoa leptomitiformis]. / Vydelenie,ochistka i svoistva malatdegidrogenazy iz serobakterii Beggiatoa leptomitiformis.

Malate dehydrogenase (E.C. 1.1.1.37) from bacterium Beggiatoa leptomitiformis was isolated and purified 123 times using a five-step purification procedure including the enzyme extraction, ammonium sulfate protein fractionation, gel filtration, ion exchange chromatography, and gel chromatography. The enzyme was homogenous according to the electrophoresis data; its activity was 20.43 U/mg proteins. This malate dehydrogenase is a homotetramer (Mr = 172 kDa). The catalytic and thermodynamic properties, as well as the analysis of the published data suggest that the tetrameric structure of the enzyme allows it to participate in constructive metabolism supplying the cell with organic acids as a source of carbon.

Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa.

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.

[Dependence of the structure of malate dehydrogenase on the type of metabolism in fresh water filamentous colorless sulfur bacteria of the Beggiatoa species]. / Zavistimost' struktury malatdegidrogenazy ot tipa metabolizma u presnovodnykh nitchatykh bestsvetnykh serobakterii roda Beggiatoa.

Major pathways of carbon metabolism were studied in strains D-402 and D-405 of freshwater colorless sulfur bacteria of the genus Beggiatoa grown organotrophically and mixotrophically. The bacteria were found to possess all the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles. When organotrophic growth changed to mixotrophic one, the activity of the TCA cycle enzymes decreased 2- to 3-fold, but the activity of enzymes of the glyoxylate cycle increased threefold. It follows that, in the oxidation of thiosulfate, organic compounds no longer play the leading part in the energy metabolism, and most of electrons that enter the electron transport chain (ETC) derive from inorganic sulfur compounds. A connection was established between the structure and kinetic characteristics of malate dehydrogenase--an enzyme of the TCA and glyoxylate cycles--and the type of carbon metabolism in the strains studied. Malate dehydrogenase in organotrophically grown cells of strains D-402 and D-405 is dimeric, whereas in strain D-402 grown mixotrophically it is tetrameric.

Alternative system of succinate oxidation in glyoxysomes of higher plants.

Succinate oxidation in scutella of germinating seeds of wheat and maize was investigated. Besides oxidation via succinate dehydrogenase (SDH; EC 1.3.99.1), an alternative path of succinate oxidation insensitive to SDH inhibitors--malonate and thenoyltrifluoroacetone (TTFA)--was revealed. Using isopicnic sucrose gradient it was shown that this path is localized in glyoxysomal membranes. Glyoxysomal succinate oxidase (GSO) converts succinate directly into malate with the production of hydrogen peroxide identified using auxiliary enzymes malate dehydrogenase and peroxidase. GSO is most active during the intensive operation of the glyoxylate cycle (3-5 days of germination). Quinacrine, the inhibitor of flavine-containing oxidases, strongly suppressed the activity of GSO. Km for succinate is 18 mM for GSO from maize scutellum. It is concluded that in scutella of cereal seeds the glyoxysomal succinate oxidation non-linked with ATP synthesis operates.