Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom.
Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).
J
J
Shiloah
C
C
Klibansky
A
A
de Vries
A
A
Berger
D000818:Animals; D019281:Dimerization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008244:Lysophosphatidylcholines; D008245:Lysophospholipase / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D014757:Viper Venoms / Q000201:enzymology*
D008244:Lysophosphatidylcholines; D014757:Viper Venoms; D010741:Phospholipases A; D008245:Lysophospholipase