Lysine-49-phospholipases A2 from Trimeresurus flavoviridis venom are membrane-acting enzymes.
Basic proteins I and II (BP-I and BP-II) isolated from the venom of Trimeresurus flavoviridis (Habu snake) are isozymes of highly active Asp-49 phospholipase A2 (Asp-49-PLA2) and classified into the group Lys-49-PLA2. BP-II was found to elicit a strong contraction of guinea pig ileum, and this activity was inhibited completely by 1 microM indomethacin, an inhibitor of the arachidonate cascade. BP-II was inactive in the Ca(2+)-free medium, and p-bromphenacylated His-48-BP-II was also inactive. BP-II exhibited no binding affinity for the cells expressing PLA2 receptors. These results indicated that the contraction elicited by BP-II is due to the hydrolytic action of BP-II, liberating arachidonic acid from the ileum phospholipid biomembranes. In spite of its limited lipolytic activities (av. 0.9% of Asp-49-PLA2) against monomers and micelles of synthetic phospholipids, BP-II hydrolyzed considerably strongly the phospholipids in the artificial bilayer vesicles. Arachidonic acid released from liposomes of beta-arachidonoyl-gamma-stearoyl-L-alpha-phosphatidylcholine was determined by HPLC, and the activity of BP-II was estimated to be about 75% as compared to Asp-49-PLA2. Liposomes encapsulating carboxyfluorescein exhibited a strong dye-leakage induced by BP-II in a concentration-dependent manner, only in the Ca(2+)-containing buffer. The net result from all these observations was that BP-II, a Lys-49-PLA2, is an enzyme that hydrolyzes the membrane phospholipids. In contrast to BP-II, BP-I was found to be considerably weak in hydrolyzing membrane phospholipids, although its activities were distinct. BP-I and BP-II share a common sequence with the sole exception 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-PLA2s is the residue required for discriminatory recognition of beta-arachidonoyl-phospholipid membranes.
Shimohigashi
Y
Y
Department of Chemistry, Kyushu University, Fukuoka.
Tani
A
A
Matsumoto
H
H
Nakashima
K
K
Yamaguchi
Y
Y
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D003435:Crotalid Venoms; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D007527:Isoenzymes / Q000302:isolation & purification / Q000494:pharmacology*; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D054467:Phospholipases A2; D011506:Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000378:metabolism; D054507:Receptors, Phospholipase A2
D003435:Crotalid Venoms; D016861:Cyclooxygenase Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D008051:Lipid Bilayers; D008563:Membrane Lipids; D011506:Proteins; D011956:Receptors, Cell Surface; D054507:Receptors, Phospholipase A2; C029748:Trimeresurus venoms; C063854:basic protein I; D016718:Arachidonic Acid; C024098:6-carboxyfluorescein; D010741:Phospholipases A; D054467:Phospholipases A2