LYSINE-49-PHOSPHOLIPASES A(2) FROM TRIMERESURUS-FLAVOVIRIDIS VENOM ARE MEMBRANE-ACTING ENZYMES

Basic proteins I and II (BP-I and BP-II) isolated from the venom of Tr imeresurus flavoviridis (Habu snake) are isozymes of highly active Asp -49-phospholipase A(2) (Asp-49-PLA(2)) and classified into the group L ys-49-PLA(2). BP-II was found to elicit a strong contraction of guinea pig ileum, and this activity was inhibited completely by 1 mu M indom ethacin, an inhibitor of the arachidonate cascade. BP-II was inactive in the Ca2+-free medium, and p-bromophenacylated His-48-BP-II was also inactive, BP-II exhibited no binding affinity for the cells expressin g PLA(2) receptors. These results indicated that the contraction elici ted by BP-II is due to the hydrolytic action of BP-II, liberating arac hidonic acid from the ileum phospholipid biomembranes, In spite of its limited lipolytic activities (av. 0.9% of Asp-49-PLA(2)) against mono mers and micelles of synthetic phospholipids, BP-II hydrolyzed conside rably strongly the phospholipids in the artificial bilayer vesicles. A rachidonic acid released from liposomes of idonoyl-gamma-stearoyl-L-al pha-phosphatidylcholine was determined by HPLC, and the activity of BP -II was estimated to be about 75% as compared to Asp-49-PLA(2). Liposo mes encapsulating carboxyfluorescein exhibited a strong dye-leakage in duced by BP-II in a concentration-dependent manner, only in the Ca2+-c ontaining buffer. The net result from all these observations was that BP-II, a Lys-49-PLA(2), is an enzyme that hydrolyzes the membrane phos pholipids. In contrast to BP-II, BP-I was found to be considerably wea k in hydrolyzing membrane phospholipids, although its activities were distinct, BP-I and BP-II share a common sequence with the sole excepti on of Asp-67 (BP-I) and Asn-67 (BP-II) in the aligned sequences, This implies that the amino acid at position 67 of Lys-49-PLA(2)s is the re sidue required for discriminatory recognition of beta-arachidonoyl-pho spholipid membranes.