Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.