[Activation of calcium transport in synaptosomes by phosphatidylinositol-specific phospholipase C]. / Aktivatsiia transporta kal'tsiia v sinaptosomakh fosfolipazoi C, spetsifichnoi k fosfatidilinozitam.

The isotope labeling method was used to study the influence of phospholipases C of different origin and specificity on Ca2+ accumulation in rat brain synaptosomes. It was found that phospholipases C specific to phosphatidylinositides (PI) stimulate Ca2+ transport into synaptosomes, while non-specific phospholipase C, which hydrolyzes different membrane lipid fractions, decreases the Ca2+ content in synaptosomes. It is supposed that the stimulating effect of PI-specific phospholipases C is determined by the activation of PI metabolism, which results in an increase in the content of some PI metabolism products serving as Ca2+ ionophores in synaptosomal membranes. The inhibition of Ca2+ uptake by synaptosomes treated with non-specific phospholipase C is thought to result from partial disruption of synaptosomal membranes.

[Properties of the phospholipases C from Bacillus cereus]. / Nekotorye svoistva fosfolipaz C iz Bacillus cereus.

Three different phospholipases C--the so-called phospholipase C which hydrolyses phosphatidylcholine (Pch-PLC), sphingomyelinase (SM-PLC) and phosphatidylinositol hydrolysing phospholipase C (PI-PLC)--were separated from the culture filtrate of Bacillus cereus using column chromatography on DEAE-sephadex A-50. The pI values were estimated to be 5.0 +/- 0.3 for PI-PLC and 5.3 +/- 0.2 for SM-PLC. The effect of some bivalent cations was studied. Metal ions had no effect on the activity of PI-PLC, whereas Mg2+ and Co2+ in concentrations from 1 to 5 mM highly activated SM-PLC. Mg2+ failed to activate PCh-PLC, and Co2+ even inhibited it. EDTA of o-phenantroline had a rather inhibitory effect on Pch-PLC, but they almost did not affect SM-PLC.

[Effect of the redox state of glutathione on acetate kinase activity in E. coli]. / Vliianie okislitel'no-vosstanovitel'nogo sostoianiia glutationa na aktivnost' atsetatkinazy E. coli.

Oxidized glutathione inhibits acetate kinase (EC 2.7.2.1) of E. coli. The rate of inactivation depends on ATP concentration. The rate constant for the glutathione-induced inhibition is 0.17 min-1, Ki is 4.2 mM (pH 7.2, 25 degrees C). The inhibition of acetate kinase by glutathione is reversible, the equilibrium constant being equal to 4.4 or 0.09 at saturating concentrations of ATP (pH 8.0, 25 degrees C). The physiological level of reduced and oxidized glutathione can modulate the acetate kinase activity in vivo.

[Biosynthesis of extracellular phospholipase C (lecithinase) from Bacillus cereus depending on the nutrient medium composition and pH]. / Binosintez vnekletochnoi fosfolipazy S (letsitinazy) Bacillus cereus v zavisimosti ot sostava pitatel'noi spedy i pH.

The nutrient medium to provide rapid growth of microbial cells of Bacillus cereus str. 504, and biosynthesis of extracellular phospholipase C(EC 3.1.4.3) was selected. The nutrient medium contained acidic casein hydrolyzate, yeast extract or enzymatic hydrolyzate of fodder yeast, glucose, NaCl, NaHCO3, and Na2HPO4. The activity of secreted phospholipase C reached maximum at pH 6.3--6.6.

[Acetate kinase chromatography on agarose derivatives]. / Khromatografiia atsieatkinazy na proizvodnykh agarozy.

Acetate kinase from E. coli K-12 was studied chromatographically on omega-aminoalkyl polysacharide sorbents. The dependence of the protein sorption-desorption on ionic strength and the effect of pH on the acetate kinase sorption were studied. The increase in the ionic strength caused a decrease in the amount of protein sorbed on the hexamethylenediamine- and chlorotriasinehexamethylenediamine sepharoses. On hexamethylenediamine-, octamethylenediamine- and dimethylhexamethylenediamine agaroses acetate kinase was adsorbed within the pH range of 6.5-9.0, whereas on the chlorotriasinehexamethylenediamine sepharose--at pH 6.5-8.0. The active protein was eluted at ionic strength of 0.14-0.17 M. Acetate kinase was not adsorbed on the carboxypropyonylaminohexyl sepharose within the pH range studied, i.e. 5.0-9.0 and was not adsorbed on hexamethylenediamine agarose at pH 4.0 and on chlorotriasinehexamethylenediamine sepharose--at pH 9.0. The mechanism of the enzyme-adsorbent interaction is discussed.

[Isolation and some properties of phospholipase D from Bacillus subtilis G-22]. / Vydelenie i nekotorye svoistva fosfolipazy D iz Bacillus subtilis sht. G-22.

A method of isolating highly purified phospholipase D from Bac. subtilis G-22 is described. It includes ammonium sulphate fractionation, thermal denaturation, chromatography on lipoprotein bound with sepharose 6B and AH-sepharose 4B. The enzyme is 130-fold purified, its yield exceeds 90.0%, its specific activity is 164 units per mg of protein. The homogeneity of the enzyme is demonstrated by polyacrylamide gel electrophoresis, ultracentrifugation, isoelectric focusing and N-terminal amino acid determination by means of dinitrophenylation and dancylation. Proline is found to be N-terminal amino acid. The molecular weight of the enzyme, as determined from gel filtration through Sephadex G-100, is 21500 +/- 300, its sedimentation constant is 1.4S, isoelectric point is at pH 4.2. The molecular weight calculated from amino acid composition, is 21000--22000. Polypeptide chain contains of 196--205 amino acid residues. Phospholipase D develops its maximal activity at pH 8.5 and does not contain free SH-groups. Benzylsulphofluoride does not inhibit the enzyme activity. Phospholipase D is activated by Cd2+, Co2+, Zn2+, Ca2+ and is inhibited by EDTA, pIi50 being about 2.6.

[Purification of phospholipase C from Bacillus cereus by chromatography on aminoalkylpolysaccharide adsorbents]. / Khromatograficheskaia ochistka fosfolipazy s iz Bacillus cereus na aminoalkilpolisakharidnykh sorbentakh.

Purification of phospholipase C from Bac. cereus by chromatography on aminoalkylpolysaccharide adsorbents is described. The dependence of the degree of enzyme purification on the amount of ligant and effect of pH and buffer systems on the adsorption-desorption of phospholipase have been studied. At a pH below 9.0 phospholipase C is not retained by the adsorbents and is purified 4-5-fold and up to 23-fold, when aminoalkyl-Sepharose and hexamethylenediamine Sephadex are used respectively. With an increase in the pH value up to 10.0, the enzyme is bound by the adsorbent and is eluted with a 40-90% yield of activity and 7-10-fold purification. The resulting phospholipase C is highly purified and electrophoretically homogeneous. A mechanism of the enzyme-adsorbent interaction is discussed.

[Phospholipase of Bacillus cereus]. / Fosfolipaza Bacillus cereus

Phospholipase activity of 10 strains of Bacillus cereus was studied. The most active strain of Bac. cereus--phospholipase producer was selected. A cultivation mixture of Bac. cereus optimal for the phospholipase synthesis was found to include peptone, yeast extract, glucose, NaCl and Na2HPO4. Proper conditions for the synthesis of phospholipase in flasks, 20 l and 250 l fermenters were tested. The maximum increase of the phospholipase activity occurred by the 5-9th hour of microbial growth at pH 6.0-8.0. Further cultivation, foaming, strong aeration, pH increase (over 8.0) reduced the accumulated activity. By fractionation with (NH4)2SO4, ethanol precipitation, protamine sulphate treatment with subsequent Sephadex G-100 gel filtration phospholipase (EC 3.1.4.3) was purified 300-fold from the culture liquid of Bac. cereus str. 504. The preparation was examined electrophoretically in 7% polyacrylamide gel at alkaline pH. The effect of metal salts and EDTA on phospholipase activity was studied. Thermostability, substrate specificity and pH optimum of purified phospholipase were investigated.