Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

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Cytotoxic and antitumor activity of the biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV) (VO(mal) ) was investigated in comparison with inorganic vanadium(IV) compound - vanadyl sulfate (VOSO ) and also with oxovanadium monocomplex with L-malic acid (VO(mal)) and vanadyl biscomplex with acetylacetonate. In this purpose the effect of vanadyl compounds on growth of normal human skin fibroblasts and tumor cells of different lines: mouse fibrosarcoma (L929), rat pheochromocytome (PC12), human liver carcinoma (HepG2), virus transformated mouse fibroblast (NIN 3T3), virus transformated cells of human kidney (293) were investigated. The results showed that VO(mal) was not toxic for normal human skin fibroblasts but considerably inhibited growth of cancer cells in culture. Cytotoxic antitumor effect of vanadium complexes was found to be dependent оn incubation time and concentration and on type of cells and nature of ligands of the central group of the complex (VO2+). These studies provide evidence that VO(mal) may be considered as a potential antitumor agent due to its low toxicity in non-tumor cells and significant anticancer activity.

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Hepatoprotective effect of Phospholiv, a phospholipid preparation containing phosphatidylcholine from sunflower seeds and glycyrrhizic acid trisodium salt, is studied using a model of CC14-induced cirrhosis of the liver. Phospholiv protects hepatic tissues from necrotic and dystrophic changes and prevents the development of cirrhosis. Phospholiv restores impaired RNA and protein synthes is under conditions of chronic intoxication.