Immunochemical and molecular differentiation of 43 000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses.
Synaptic membranes, highly enriched in nicotinic receptor, contain three 43 000 molecular weight (Mr) peripheral proteins (distinctive in their peptide mapping profiles and earlier designated v1, v2, and v 3) as well as the receptor alpha 2 beta gamma delta integral membrane subunits. Of the three proteins, only v1 is copurified with the membrane-bound receptor, while v2 and v3 are prominent cytosolic proteins, which are retained at significant levels in receptor-rich membranes during multistep centrifugation and affinity partitioning purification procedures [Gysin, R., Wirth, M., & Flanagan, S. D. (1981) J. Biol. Chem. 256, 11373-11376]. Peptide mapping analysis of Torpedo v3 and rabbit skeletal actin indicates that the two proteins are closely related. The enzymatic activity, creatine phosphokinase (EC 2.7.3.2), copurifies with v2 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 v1, creatine phosphokinase, and actin by the action of mild alkali, v1 remains in a high molecular weight form. Dissociation of v1 into lower molecular weight species requires urea or sodium dodecyl sulfate (NaDodSO4). Preparation of essentially pure v1 was achieved by eluting the v1 protein spots directly from naDodSO4-isoelectric focusing gels loaded with alkali extracts derived from membranes highly enriched in nicotinic receptor. Amino acid compositions of the purified fractions indicate that v1 and Torpedo creatine phosphokinase have distinct amino acid compositions from each other and from that of actin.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003402:Creatine Kinase / Q000032:analysis; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013570:Synaptic Membranes / Q000032:analysis*; D014101:Torpedo
D000199:Actins; D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D003402:Creatine Kinase