Phosphorylation of endogenous and exogenous proteins by rat liver microsomal protein kinases

Abstract

1. 1. Rat liver microsomes contain protein kinase activities which phosphorylate both endogenous and exogenous protein substrates. The endogenous reaction is independent of cyclic AMP while the exogenous one requires this nucleotide for maximum activity. 2. 2. These phosphorylations are catalyzed by different protein kinases, denoted as Type A and Type B. Type A protein kinase is firmly bound to the microsomal membrane and is responsible for phosphorylating endogenous proteins, which are also integral membrane proteins. 3. 3. Type B protein kinase is loosely associated with the membrane and phosphorylates exogenous proteins; preferring protamine > histone f 2b > histone f 1. Histones f 2a, f 3, phosvitin, bovine serum albumin, microsomal cytochrome P-450, and NADPH-cytochrome P-450 reductase were poor substrates. 4. 4. Type A protein kinase is more stable to incubation than Type B. Type B protein kinase is inhibited by imidazole, but type A enzyme is not. 5. 5. Endogenous protein phosphorylation was increased 4-fold by the addition of Triton X-100 under conditions which solubilized 78% of the membrane proteins. 6. 6. The detergent is believed to increase the accessibility of protein substrates to Type A protein kinase and to cause higher level of phosphorylation.