An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver.
1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation
D009712:Nucleotides, Cyclic; D010455:Peptides; D010716:Phosphatidylinositols; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium