CHEMICAL MODIFICATION AND INACTIVATION OF PHOSPHOLIPASES A(2) BY A MANOALIDE ANALOG

Chemical modification and inactivation of bovine pancreatic, porcine p ancreatic, Naja naja atra and Pseudechis australis phospholipases A(2) (PLA(2)s), belonging to Group I, and of Trimeresurus flavoviridis, Vi pera russelli russelli and Agkistrodon halys blomhoffii PLA(2)s, belon ging to Group II, were investigated by the use of a manoalide (MLD)-an alogue, nyl)-8,12-dimethyl-4-formyl-3,7,11-tridecatrienol. At appropri ate time intervals, residual PLA(2) activities towards monodispersed, anionic mixed micellar and non-ionic mixed micellar substrates were me asured. We tested the protective effect of micellar n-dodecylphosphoch oline (n-C12PC) on enzyme inactivation. Inactivation of pancreatic PLA (2)s (Group I) was only observed towards anionic mixed micellar substr ates. This inactivation was completely prevented by the presence of mi cellar n-C12PC. From a fragmentation study of modified bovine pancreat ic PLA(2) using lysyl endopeptidase, we speculated that Lys-56 of this enzyme was modified by MLD-analogue and that this modification was re sponsible for enzyme inactivation. Inactivation of non-pancreatic PLA( 2)s was observed towards all types of substrate, except that no signif icant inactivation of N. naja atra PLA(2) (Group I) towards monodisper sed substrate was noted. Micellar n-C12PC protected N. naja atra PLA(2 ) (Group I) completely from inactivation by MLD-analogue, but had less er protective effects on P. australis PLA(2) (Group I), T. flavoviridi s and V. russelli russelli PLA(2)s (Group II). However, no significant protection of A. halys blomhoffii PLA(2) (Group II) activity was obse rved. These results indicate that the inactivation of pancreatic and N . naja atra PLA(2)s originates from the modification of Lys residues a t the interfacial recognition site, and that inactivation of P. austra lis, T. flavoviridis and V. russelli PLA(2)s arises from the modificat ion of Lys residues at the catalytic site, interfacial recognition sit e and regions outside both sites. The inactivation of A. halys blomhof fii PLA(2) was assumed to be due to the modification of Lys residues o utside the two sites described above.