Rat liver cytosol phosphoprotein: Purification and enzymatic phosphorylation and dephosphorylation

A phosphoprotein fraction containing both phosphorylserine and phosphoryl threonine has been isolated from rat liver cytosol and purified up to 100-fold by DEAE-cellulose column chromatography followed by gel filtration through Sephadex G-200 and G-100. The purified phosphoprotein, whose alkali labile phosphate content approximates 9 μg per mg protein, is resolved into at least five phosphorylated bands upon electrophoresis on cellulose-acetate strips, pH 7.2. The phosphorylation of cytosol phosphoprotein by [32P]ATP is catalyzed by both cytosol and microsomal phosvitin kinase, free of any protamine kinase activity. A partial dephosphorylation of the 32P-labelled phosphoprotein can be accomplished through the reversal of the protein kinase reaction, using both ADP and GDP as phosphate acceptors. An almost complete release of previously incorporated 32P from the protein is catalyzed by a mitochondrial protein phosphatase. In contrast, no dephosphorylation could be observed with crude preparations of cytosol protein phosphatase. © 1971.