Phosphorylation in vitro of Membrane Fragments from Torpedo marmorata Electric Organ. Effect on Membrane Solubilization by Detergents

Abstract

Acetylcholine receptor-rich membrane fragments purified from T. marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40,000-Mr [molecular ratio] chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent MW 43,000, 50,000 and 66,000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these 3 peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was stimulated up to 3-5-fold by K+. The effect of Na+ ions appeared less specific since Na+ reduced the labeling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43,000-Mr, 50,000-Mr and 66,000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40,000-Mr and 43,000-Mr chains. The microheterogeneity of the 43,000-Mr chain, but not that of the 40,000-Mr chain, might result from a selective phosphorylation of this particular chain.