Characterization of three edema-inducing phospholipase A 2 enzymes from habu ( Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid

Abstract

A basic phospholipase A 2 (PLA 2) enzyme, TFV PL-X (pI 9.2) and two acidic PLA 2 enzymes, TFV PL-Ia (pI 4.9) and TFV PL-Ib (pI 4.5) were purified from Trimeresurus flavoridis venom on CM-Sephadex C-25 and QAE-Sephadex A-25 columns, respectively. The basic enzyme exists as a monomer, whereas the acidic enzymes are dimers. These enzymes differ in properties such as molecular weight, K m , optimum pH and temperature and pharmacological properties. The basic enzyme hydrolysed purified phospholipids in the order of PC > PE > PS > PI = 0, while for TFV PL-Ia and TFV PL-Ib the order was PC > PE > PS = PI = 0. TFV PL-X was comparatively more toxic, with an ld 50 value of 4.2 μg/g (i.p.), while the acidic PLA 2 enzymes had ld 50 values above 8 μg/g (i.p.). All three enzymes induced edema when injected into the mouse foot pad. Aristolochic acid, an alkaloid (8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid) from the medicinal plant Aristolochia radix, interacts with these PLA 2 enzymes. It is a competitive inhibitor with varying affinity when PC is used as substrate. Aristolochic acid inhibits direct and indirect hemolytic activity, as well as edema-inducing activity, of TFV PL-X, but fails to neutralize the lethal potentcy of the enzyme. On the other hand, it inhibits direct and indirect lytic activity of TFV PL-Ia and TFV PL-Ib only at 10-fold higher concentrations and it enhances the edema-inducing activity of these enzymes. Such effects of aristolochic acid indicates that (1) different mechanisms may be involved in the edema-inducing activity of PLA 2 enzymes and (2) catalytic and pharmacological sites are separate on the PLA 2 molecule.