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Journal of Lipid Research, Vol. 14, 267-278, May 1973
Copyright © 1973 by Lipid Research, Inc.

Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom

J. Shiloah , Chaya Klibansky , A. de Vries , and A. Berger

The Rogoff-Wellcome Medical Research Institute, Tel Aviv University and Beilinson Medical Center, Petah Tikva, and The Weizmann Institute of Science, Rehovoth, Israel

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°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).

Supplementary key words lecithin • lysolecithin • 2-deoxylysolecithin • lysophospholipase • micellar structure

Submitted on March 1, 1971
Revised on August 21, 1972
Accepted on December 18, 1972

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