Polyamine Stimulation of Phosphorylation of Nonhistone Acidic Protein in Nuclei and Nucleoli from Physarum polycephalum

Intact isolated nuclei and nucleoli from the slime mold Physarum polycephalum incorporated inorganic [32P]phosphate into the phenol‐soluble acidic nuclear proteins when incubated for 1 h in a phosphorylation mixture consisting of [32P]phosphate (pH 6.8), glucose, Mg2+, and ATP. The polyamines spermidine and spermine, or equimolar combinations of these compounds with putrescine, at 1 mM total amine concentration, stimulated phosphorylation of these nuclear proteins compared to control experiments lacking these compounds. The magnitude of enhanced phosphorylation ranged from 6‐fold to 30‐fold depending on the combination of polyamines employed and the given nuclear preparation. Magnesium ion could not substitute for the polyamines in effecting this stimulation. Fractionation of the phosphorylated proteins by polyacrylamide gel electrophoresis in sodium dodecylsulfate revealed that different polypeptides among them were phosphorylated by nuclei in the presence, versus the absence, of the polyamines. In the presence of all three polyamines, each at 0.33 mM, enhanced phosphorylation was primarily due to [32P]‐phosphate incorporation into four major nuclear proteins. These proteins had molecular weights of 14000, 27000, 52000, and 70000, respectively. In the absence of the polyamines, three major proteins were phosphorylated with molecular weights of 17500, 21200, and 25000, respectively. The polypeptides of 52000 and 70000 molecular weight were also selectively phosphorylated in, and isolated from, intact nucleoli after incubation in phosphorylation mixtures supplemented with 1 mM total polyamines. The [32P]phospho‐protein linkage of the 32P‐containing substances in polyacrylamide gels was established by the capacity of pronase to eliminate these protein bands and by their insensitivity to nuclease digestion. Polyamine stimulation of the phosphorylation of these nuclear proteins was unaltered by 2‐propynylamine, an irreversible inhibitor of amine oxidase. Moreover, H2O2 did not stimulate phosphorylation of these proteins by intact nuclei in which amine oxidase was inhibited by 2‐propynylamine.