Immunochemical and molecular differentiation of 43,000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses

Abstract

Synaptic membranes, highly enriched in nicotinic receptor, contain 3 43,000 MW peripheral proteins (distinctive in their peptide mapping profiles and earlier designated .nu.1, .nu.2, and .nu.3), as well as the receptor .alpha.2.beta..gamma..delta. integral membrane subunits. Of the 3 proteins, only .nu.1 is copurified with the membrane-bound receptor, while .nu.2 and .nu.3 are prominent cytosolic proteins; these are retained at significant levels in receptor-rich membranes during multistep centrifugation and affinity partitioning purification procedures [Gysin, Wirth, et al. 1981]. Peptide mapping analysis of Torpedo .nu.3 and rabbit skeletal actin indicates that the 2 proteins are closely related. The enzymatic activity, creatine phosphokinase (EC 2.7.3.2), copurifies with .nu.2 during chromatofocusing fractionation of the cytosol. The Torpedo electroplax form of creatine phosphokinase has an electrophoretic mobility identical with that of the mammalian skeletal muscle form of the enzyme. Upon release of the membrane-bound forms of .nu.1, creatine phosphokinase and actin by the action of mild alkali, .nu.1 remains in a high MW form. Dissociation of .nu.1 into lower MW species requires urea or sodium dodecyl sulfate [SDS]. Preparation of essentially pure .nu.1 was achieved by eluting the .nu.1 protein spots directly from SDS4-isoelectric focusing gels loaded with alkali extracts derived from membranes highly enriched in nicotinic receptor. Amino acid compositions of the purified fractions indicate that .nu.1 and Torpedo creatine phosphokinase have distinct amino acid compositions from each other and from that of actin. To determine whether the observed differences in the peripheral protein peptide mapping and amino acid composition profiles are reflected as well in their antigenic properties, an antiserum against an electrophoretically purified fraction highly enriched in .nu.1 was prepared. This antiserum is essentially monospecific for .nu.1, detecting as little as 15 ng of .nu.1 present in alkali extracts of affinity-purified membranes. The immunochemical analysis serves to emphasize the conclusion that, although .nu.1 overlaps in both MW and isoelectric focusing parameters with .nu.2 (creatine phosphokinase) and .nu.3 (Torpedo actin), they display no evolutionary interrelationships, having distinct antigenic sites, solubility properties and amino acid compositions.