Phosphorylation of nuclear proteins from rabbit cerebrum, cerebellum and liver in vitro.

Abstract

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ‐32P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [32P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14m‐NaCl‐soluble, 0.25 n‐HCl‐soluble (mainly histone) and acidic phenol‐soluble proteins, NaCl‐soluble protein showed the highest phosphorylation while HCl‐soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol‐soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ‐32P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.