Phosphorylation of Proteins in Rat Liver.

Smooth and rough endoplasmic reticulum and free ribosomes from rat liver each show cyclicAMP-stimulated protein kinase activity utilizing exogenous substrates. The protein kinases of smooth and rough endoplasmic reticulum can be divided into two classes, one which is extracted by ionic media, and one more firmly attached enzyme fraction which is solubilized by Triton X-100. The protein kinase of free ribosomes is extracted by ionic media. All the three microsomal fractions support an endogenous phosphorylation of proteins which is only slightly stimulated by cyclic AMP. The endogenous phosphorylation shows two pH optima at pH 6.5 and 8.5. The phosphate is incorporated into seryl and threonyl residues of several protein species. Two major phosphoproteins are present in both smooth and rough endoplasmic reticulum, while a third major phosphoprotein is present only in the smooth fraction. There are also several minor phosphoproteins in the two fractions. The endogenous phosphorylation is initially rapid, especially in smooth and rough endoplasmic reticulum where it reaches a maximum after 10—15 rain incubation. The endogenously phosphorylated microsomal fractions also support an endogenous dephosphorylation, which is rapid initially, but which leaves approximately 60% of the phosphoryl groups unhydrolyzed. Like the protein kinase the proteins of smooth and rough endoplasmic reticulum which can undergo endogenous phosphorylation can be divided into two classes, one which is extracted in ionic media and one more tightly bound which is solubilized by Triton X-100. Protein kinase, cyclic-AMP-binding material and protein substrates extracted from smooth and ruogh endoplasmic reticulum by salt or detergent all show recoveries substantially exceeding 100%, suggesting that these activities are partly masked while associated with membrane material. [ABSTRACT FROM AUTHOR]