Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents.
Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling 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.
D000109:Acetylcholine / Q000378:metabolism; D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism; D008970:Molecular Weight; D010276:Parasympatholytics / Q000494:pharmacology; D010277:Parasympathomimetics / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic; D012967:Sodium Dodecyl Sulfate
D000779:Anesthetics, Local; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003902:Detergents; D010276:Parasympatholytics; D010277:Parasympathomimetics; D011950:Receptors, Cholinergic; D012967:Sodium Dodecyl Sulfate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D000109:Acetylcholine