CONVERSION OF BOVINE PANCREATIC PHOSPHOLIPASE A(2) AT A SINGLE-SITE INTO A COMPETITOR OF NEUROTOXIC PHOSPHOLIPASES A(2) BY SITE-DIRECTED MUTAGENESIS

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A(2) variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng , M. C. (1991) Biochemistry 30, 11473-11477), The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A(2) generated by site-dire cted mutagenesis of Phe(22) of the wild-type enzyme to Tyr, The IC50 o f this inhibition was estimated to be 1 mu M. In sharp contrast, the w ild-type enzyme gave no effect even at 50 mu M. This mutation resulted in only minor and localized structural perturbations with little effe ct on enzymatic activity. Other phospholipase A(2) molecules capable o f competing with crotoxin for this binding invariably have Tyr at this position, It was concluded that this Tyr residue is an important dete rminant for the binding of a number of phospholipase A(2) variants to the 45-kDa receptor.