[Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase].

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K(M) value for L-asparagine is 17 +/- 0.9 microM, the optimal temperature is 60 degrees C, pH is 8.0, pI is 5.4 +/- 0.3, the L-glutaminase activity is no more than 5-6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.

[Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis].

The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using several tumor cells lines: Jurkat and Molt-4 (human T-lymphoblastic leukemia), MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E. coli L-asparaginase produced by "Medak" (Germany) was used as a reference. The cell growth inhibition data indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. These results allow us to conclude that this L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.

[Purification and properties of recombinant Erwinia carotovora L-asparaginase expressed in E.coli cells]. / Vydelenie, ochistka i nekotorye svoistva rekombinantnoi L-asparaginazy Erwinia carotovora, ékspressirovannoi v kletkakh E. coli.

The method of purification Erwinia carotovora recombinant L-asparaginase, expressed in E.coli, including ultrasonic disintegration of biomass, fractionation ammonium sulfate and column chromatography on CM- or SP-Sepharose has been developed. According to SDS-PAAGE the enzyme preparation was homogeneous, its specific activity and yield consist respectively about 620 IU/mg of protein and 75%. Physical-chemical and structural properties of recombinant Erwinia carotovora L-asparaginase are similar to the enzymes from the wild strains Erwinia carotovora and recombinant L-asparaginase Erwinia chrysanthemi.

[Isolation and purification of recombinant truncated (delta2-27) cytochrome P450 2B4 expressed in E. coli cells as a fusion protein with glutathione-S-transferase]. / Vydelenie i ochistka rekombinantnogo "usechennogo" (delta2-27) tsitokhroma P450 2B4, ékspressirovannogo v kletkakh E. coli v sostave gibridnogo belka s glutation-S-transferazoi.

This paper describes the modification of the method by Coon and Pernecky (Meth. Enzymol. 1996, 272, 25-34) for purification of truncated (delta 2-27) recombinant form of cytochrome P450 2B4 expressed in E. coli as fusion protein with glutathione-S-transferase. The modifications included optimisation of conditions for proteolytic reaction of fusion protein with thrombine, removal of this protease from purified cytochrome P450 preparations using column chromatography on hydroxyapatite, introduction of the additional step for obtaining of spheroplasts using of lysozyme, and optimisation of conditions for enzyme stabilisation during of its purification and storage. The overall yield of purified cytochrome was 20% and the specific content of P450 was 14,5 nmol/mg protein was measured. This method is suitable for large-scale isolation of high purified cytochromes P450 which are necessary for study of structure-functional relationships of this hemoprotein with protein partners as well as for investigation of its structure and mechanism of action.

[Isolation and specificity of novel restriction endonucleases Bsp40091 and AsiI, isoschizomers of BamHI]. / Vydelenie i opredelenie spetsifichnosti novykh éndonukleaz restriktsii Bsp40091 i AsiI izoshizomerov BamHI.

New type II restriction endonucleases AsiI and Bsp40091 are detected in Azotobacter species N55 and Bacillus species 4009, respectively. Purified preparations of the restriction enzymes free from interfering nucleases and phosphatases were obtained by column chromatography on phosphocellulose and heparin-sepharose (Asil) and phosphocellulose and DEAE-cellulose (Bsp40091). The yield of purified AsiI and Bsp40091 was 16 x 10(3) and 8 x 10(3) units per g of wet cells, respectively. The above restriction endonucleases recognize the 5'-G decreases GATCC-3' sequence on double-stranded DNA and cleave it as shown, thus being true isoschizomers of BamHI restriction endonuclease.

[Site-specific BcuAI endonuclease from Bacillus cereus A]. / Sait-spetsificheskaia éndonuleaza BcuAI iz Bacillus cereus A.

A new restriction endonuclease was isolated from the Bacillus cereus BKM B-814 by means of the cell disruption with ultrasonication, ammonium sulfate fractionation of the cell-free extract, and chromatography on DEAE-Sepharose to give about 1400 U of the enzyme per gram of cells. The enzyme revealed the maximum activity at 30-37 degrees C, pH 7.6-8.2, and 5-10 mM MgCl2 under a high ionic strength (50 mM Tris-HCl, 100 mM NaCl). The site-specific endonuclease BcuAI was found to recognize the 5' G decreases G(A/T)CC sequence in double-stranded DNA and cleave it as shown with the arrow, thus being a true isoschisomer of the AvaII restriction endonuclease.