An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver

Abstract

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 [γ-32P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [γ-32P]ATP was markedly stimulated by Mg2+, but not by cyclic AMP, cyclic GMP or Ca2+. The phosphorylation of certain polypeptides was preferentially stimulated by Mg2+. Addition of cyclic AMP resulted in a decrease in the amount of 32P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [γ-32P]GTP was found to be a relatively poor donor of 32P as compared with [γ-32P]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 [γ-32P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of 32P from [γ-32P]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 32P into this lipid was also markedly stimulated by Mg2+. 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.