Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A2s on brain synaptic membranes.
Crotoxin and other neurotoxic phospholipase A2s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several others to synaptic membranes has been demonstrated previously. In this study we used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After 125I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of approximately 60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein of approximately 45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of around 1 x 10(-8) M. Mojave toxin and some other phospholipase A2s were also highly inhibitory to this conjugation, and notexin and others were less effective, while beta-bungarotoxin and pancreatic PLA2 were totally ineffective. We concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A2s.
Yen
C H
CH
Institute of Biochemical Sciences, National Taiwan University, Republic of China.
Tzeng
M C
MC
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003439:Crotoxin / Q000378:metabolism*; D006168:Guinea Pigs; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013570:Synaptic Membranes / Q000378:metabolism*
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2