[Isolation and some properties of phospholipase D from Bacillus subtilis 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.
D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology*; D002413:Cations, Divalent; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002413:Cations, Divalent; D010740:Phospholipases
Vydelenie i nekotorye svoistva fosfolipazy D iz Bacillus subtilis sht. G-22.