Abstract
A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A2 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 A2 generated by site-directed mutagenesis of Phe22 of the wild-type enzyme to Tyr. The IC50 of this inhibition was estimated to be 1 microM. In sharp contrast, the wild-type enzyme gave no effect even at 50 microM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A2 molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A2 variants to the 45-kDa receptor.
Highlights
Transmitters, though most of them produce postsynaptic toxicity and other effects
As one of our approaches to understanding the structural basis for this binding, we converted bovine pancreatic PLAa, which showed no detectable binding to the synaptic membrane, into a mutant capable of competing for the binding ofcrotoxin to this receptor by site-directed mutagenesis
Mter 1251-crotoxin had been incubated with the synaptic membrane fraction from the brain to reach maximal binding, disuccinimidyl suberate or disuccinimidyl dithiobis(propionate) was employed to cross-link the binding complexes
Summary
Materials-Disuceinimidyl suberate and disuccinimidyl dithiobis(propionate) were obtained from Pierce. Generation and Purification of Mutant PLA2-Mutations of bovine pancreatic PLA, were generated by site-directed mutagenesis of a chemically synthesized gene for the wild-type protein [25] with an Amersham kit making use of the phosphorothioate method [24]. After centrifugation at 15,000 X g for 15 min, the membrane pellet was solubilized with 0.1 M Tris-HCl buffer, pH 6.8, containing 5% glycerol and 2% SDS, and analyzed by SDS-polyacrylamide gel electrophoresis [31]. Other details are as described previously [35]
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