Conversion of bovine pancreatic phospholipase A2 at a single site into a competitor of neurotoxic phospholipases A2 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 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.
Tzeng
M C
MC
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China.
Yen
C H
CH
Hseu
M J
MJ
Dupureur
C M
CM
Tsai
M D
MD
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain; D002417:Cattle; D003439:Crotoxin / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011956:Receptors, Cell Surface / Q000378:metabolism; D054507:Receptors, Phospholipase A2; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013570:Synaptic Membranes / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D017931:DNA Primers; D011956:Receptors, Cell Surface; D054507:Receptors, Phospholipase A2; D014443:Tyrosine; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2