Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.
When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009186:Myelin Sheath / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D010774:Phosvitin / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D006656:Protamine Kinase / Q000378:metabolism; D011479:Protamines / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012584:Sciatic Nerve / Q000378:metabolism*
D002364:Caseins; D011479:Protamines; D047428:Protein Kinase Inhibitors; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D010774:Phosvitin; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D006152:Cyclic GMP; D008274:Magnesium