Discriminatory recognition of membrane phospholipids by lysine-49-phospholipases A2 from Trimeresurus flavoviridis venom.
Basic proteins I and II (BP-I and BP-II) isolated from Trimeresurus flavoviridis venom, which are classified into a group of lysine-49-phospholipases A2 (Lys-49-PLA2), exhibited only limited lipolytic activity for the mixed micelles of various phospholipids. Based on the finding that BP-II elicits a strong contraction of guinea pig ileum due to the release of arachidonic acid, BP-II together with BP-I has been tested for their interaction with artificial phospholipid bilayer membranes. The dye leakage experiments indicated that BP-II interacts strongly with liposomes of beta-arachidonoyl-gamma-stearoyl-L-alpha-phosphatidylcholine. The perturbation of liposomes was observed only in the Ca(2+)-containing buffer, and as demonstrated by HPLC analyses, accompanied by the release of arachidonic acid. The concentration of Ca2+ which gave a half maximal activity of BP-II was 3.0 x 10(-4) M, suggesting that the affinity of BP-II for Ca2+ is more than 10 times stronger than that of BP-II without liposomes. These observations clearly show that Lys-49-PLA2 of BP-II is the enzyme responsible for the hydrolysis of membrane phospholipids and that Ca2+ is essential for such enzymatic activity. The interaction of BP-I with liposomes was much weaker than BP-II BP-I and BP-II share a common sequence except for 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 phospholipid membranes.
Shimohigashi
Y
Y
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
Tani
A
A
Yamaguchi
Y
Y
Ogawa
T
T
Ohno
M
M
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D003435:Crotalid Venoms / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008823:Micelles; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D017841:Trimeresurus
D003435:Crotalid Venoms; D005452:Fluoresceins; D007527:Isoenzymes; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D011506:Proteins; C029748:Trimeresurus venoms; C063854:basic protein I; D016718:Arachidonic Acid; C024098:6-carboxyfluorescein; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine