The effect of phytoprotectors on the functioning of liver NAD⁺- and NADP⁺-maliс enzymes in rats with alloxan diabetes.

The development of experimental alloxan diabetes in rats was accompanied by the increase the activity of liver NAD⁺- and NADP⁺-dependent malic enzymes (ME; NAD⁺-ME, EC 1.1.1.39 and NADP⁺-ME, 1.1.1.40) associated with an increase in the rate of transcription of genes encoding these enzymes. Oral administration of aqueous extracts of Jerusalem artichoke and olive to diabetic rats caused a noticeable decrease in blood glucose, a decrease in the rate of transcription of the studied genes; and a decrease in ME activity towards normal values. Thus, extracts of Jerusalem artichoke and olive can be used as additives to the standard therapy of diabetes mellitus.

[Molecular and biochemical studies of succinate dehydrogenase in rat liver under conditions of alloxan diabetes]. / Molekuliarnye i biokhimicheskie issledovaniia suktsinatdegidrogenazy v pecheni krys v usloviiakh alloksanovogo diabeta.

Experimental alloxan diabetes in rats causes an increase in the activity of liver succinate dehydrogenase (SDH) without changes in its isozyme composition. The observed increase in the catalytic activity of SDH clearly correlates with the intensification of transcription of the genes encoding catalytic dimer of SDH. Analysis of the methyl status of the promoters of the genes, encoding the catalytic dimer of SDH in rats under normal and experimental conditions did not reveal any dependence on the level of their expression. The obtained results of bisulfite sequencing indicate a passive role of the epigenetic mechanism of regulation of SDH gene expression in the development of alloxan diabetes. The transcription factor CREB, responsible for of gluconeogenesis in diabetes, may play an important role in the control of the transcriptional activity of the sdha and sdhb genes.

[Expression levels and activity of rat liver lactate dehydrogenase isoenzymes in alloxan diabetes]. / Urovni ékspressii i aktivnost' izofermentov laktatdegidrogenazy pecheni krys pri alloksanovom diabete.

A significant decrease in the activity of lactate dehydrogenase (LDH, EC 1.1.1.27) in liver cells of rats with alloxan diabetes was found due to a decrease in the expression of the corresponding genes. The decrease in the activity of the enzyme under study in experimental type I diabetes was associated with inactivation of the cytoplasmic isoform of LDH. It was found that the level of ldha and ldhb gene transcripts in the liver of healthy rats was higher than in animals with alloxan diabetes. The ldha gene expression demonstrated almost 9-fold decrease, while a decrease in the ldhb gene expression was less pronounced (just 1.25-fold). Probably, the decrease in the rate of functioning of the enzyme under study is associated with a decrease in the intensity of glucose uptake by cells, which leads to inhibition of glycolysis and intensification of all stages of gluconeogenesis, particularly, reversed glycolysis reactions. Thus, the data obtained by us indicate an important role of LDH in the adaptive response of cellular metabolism in the development of type I diabetes mellitus.

[Effect of jerusalem artichoke extract on the functioning of malate dehydrogenase in the liver of rats with alloxan diabetes]. / Vliianie ékstrakta topinambura na funktsionirovanie malatdegidrogenazy v pecheni krys pri alloksanovom diabete.

An increase in the activity and the appearance of a new isoform of NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) has been detected in the liver of rats with alloxan diabetes was revealed. This confirms the possibility of MDH involvement in the adaptive reaction of the body under oxidative tress caused by biochemical changes in diabetic animals. The increase in the hepatic MDH activity in rats with experimental type I diabetes mellitus (T1DM) is associated with the formation of an additional MDH isoform in peroxisomes. Data on the expression of the MDH encoding genes mdh1 and mdh2 confirm that in T1DM the increase in MDH activity occurs at the level of transcription of MDH encoding genes. The use of the extract of Helianthus tuberosus led to a marked decrease in the blood glucose concentration of rats with alloxan diabetes, abolished by the change in transcriptional activity of the studied genes and blocked the formation of new MDH isoforms in rats with experimental alloxan diabetes. This suggest that extract of H. tuberosus may be of considerable interest from the point of view of pharmacological correction of metabolic changes during the development of pathologies of this kind.

[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 and catalytic properties of NAD+- dependent malate dehydrogenase isoforms from maize mesophyll0.

Malate dehyrogenase isoforms (46- and 70-fold purifications) with specific activities of the 640 and 990 U/mg protein were obtained in an electrophoretically homogeneous state from maize mesophyll. The physicochemical and catalytic properties of these isoforms were studied. 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 SDS-PAGE demonstrated that malate dehydrogenase isoforms have an oligomeric structure comprised of identical subunits. The first isoform with a molecular weight of 126.58 kDa is tetramer, and the second isoform with a molecular weight of 63.3 is dimer.

[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.

[Characteristics of functioning of succinate dehydrogenase from flight muscles of the bumblebee Bombus terrestris (L.)].

It was found that the succinate oxidation rate in mitochondria of flight muscles of Bombus terrestris L. in- creased by a factor of 2.15 after flying for 1 h. An electrophoretically homogenous preparation of succinate dehydrogenase with a specific activity of 7.14 U/mg protein and 81.55-fold purity was isolated from B. terrestris flight muscles. It is shown that this enzyme is represented in the muscle tissue by only one isoform with R,f = 0.24. The molecular weight of the native molecule and its subunits A and B was determined. The kinetic characteristics ofsuccinate dehydrogenase (Km = 0.33 mM) and the optimal concentration of hydrogen ions (pH 7.8) were established, and the effect of salts on the enzyme activity was studied. The role of succinate as a respiratory substrate in stress and the structural and functional characteristics of the succinate dehydrogenase system in the flight muscles of insects are discussed.

[Obtaining homogenous preparations of succinate dehydrogenase isoforms from the D-507 strain of Sphaerotilus natans].

Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heteromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K(m) values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl- ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.

[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.

[Involvement of hydrogen peroxide in the regulation of coexpression of alternative oxidase and rotenone-insensitive NADH dehydrogenase in tomato leaves and calluses].

The involvement of active oxygen forms in the regulation of the expression of mitochondrial respiratory chain components, which are not related to energy storing, has been in vitro and in vivo studied in Lycopersicum esculentum L. The highest level of transcription of genes encoding alternative oxidase and NADH dehydrogenase has been observed in green tomato leaves. It has been shown that even low H2O2 concentrations activate both aoxlalpha and ndb1 genes, encoding alternative oxidase and external mitochondrial rotenone-insensitive NADH dehydrogenase, respectively. According to our results, in the case of an oxidative stress, alternative oxidase and NADH dehydrogenase are coexpressed in tomato plant tissues, and active oxygen forms serve as the secondary messengers of their coexpression.

[Role of differential expression of sdh1-1 and sdh1-2 genes in alteration of isoenzyme composition of succinate dehydrogenase in germinating maize seeds].

A probable mechanism of alteration of the isoenzyme composition of succinate dehydrogenase (SDH) due to differential expression of genes encoding subunit A was considered. The alteration of SDH activity during maize seed germination was investigated, and its maximal activity on day 4-5 of germination was found. The alteration of the sdh1-1 and sdh1-2 gene expression level during maize seed germination was evaluated using the quantitative polymerase chain reaction method. The presence of four forms of the studied enzymes, providing multiple SDH functions was found in maize inflorescence using electrophoresis in polyacrylamide gel.

[Preparation and properties of isocitrate lyase isoforms from the cotyledons of Glycine max L].

A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays.

Features of structural organization and expression regulation of malate dehydrogenase isoforms from Rhodobacter sphaeroides strain 2R.

Two isoforms of malate dehydrogenase (MDH), dimeric and tetrameric, have been found in the purple non-sulfur bacterium Rhodobacter sphaeroides strain 2R, devoid of the glyoxylate shunt, which assimilate acetate via the citramalate cycle. Inhibitory analysis showed that the 74-kDa protein is involved in tricarboxylic acid cycle, while the 148-kDa MDH takes part in the citramalate pathway. A single gene encoding synthesis of the isologous subunits of the MDH isoforms was found during molecular-biological investigations. The appearance in the studied bacterium of the tetrameric MDH isoform during growth in the presence of acetate is probably due to the increased level of mdh gene expression, revealed by the real-time PCR, the product of which in cooperation with the citramalate cycle enzymes plays an important role in acetate assimilation.

[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.

Physicochemical and kinetic characteristics of isoforms of isocitrate lyase from corn.

Three electrophoretically homogeneous isocitrate lyase (ICL) isoforms were obtained by 4-step purification from corn scutellum (ICL(1) and ICL(2)) and green leaves (ICL). Their physicochemical, kinetic, and regulatory properties were analyzed. The molecular masses of ICL(1), ICL(2) , and ICL isoforms determined by gel filtration are 164, 207, and 208 kDa, respectively. The proteins have homotetrameric quaternary structure with subunit molecular masses of 43, 48, and 47 kDa for ICL(1), ICL(2), and ICL, respectively. We found some differences in pH optimum, K(m), and regulation by divalent metal cations between ICL(1) and ICL(2) and significant similarity of ICL(2) and ICL. Based on these data, we suggest the participation of these isoforms in metabolic regulation of the glyoxylate cycle, organic acid metabolism during photorespiration in leaves and acidosis in corn seeds.

[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.

[Carbohydrate metabolism in the liver of rats in food deprivation and experimental diabetes].

In experiments with starving rats (Rattus rattus L.) and rats with alloxan-induced diabetes, the activity of enzymes and the contents of metabolites of the main metabolic pathways have been studied. It has been shown that adaptation of carbohydrate metabolism to these stress conditions has common trends: intensification of gluconeogenetic processes and glyoxylate cycle induction are observed against the background of decrease in the activities of glycolytic enzymes and the pentose phosphate pathway. A hypothetical mechanism of adaptation of rat liver cell metabolism to a deficiency of glucose as the main energy substrate is proposed.

[Isolation and purification of malate dehydrogenase isoforms from phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris].

A five-step procedure was used to obtain electrophoretically pure preparations of malate dehydrogenase (EC 1.1.1.37) from Rhodobacter sphaeroides and Rhodopseudomonas palustris. The procedure included extraction, ammonium sulfate fractionation, gel filtration, and ion exchange and gel permeation chromatography. The enzyme was found to exist in two isoforms, dimeric and tetrameric, formed by the oligomerization of identical subunits. The isoforms are assumed to be involved in different metabolic processes.

[Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants].

The metabolism of 1,4-14C-succinate and 2,3-14C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the gamma-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the gamma-aminobutyric acid shunt.