Spleen cell phosphorylation of salt soluble nuclear protein from isoproterenol treated and sorted nuclei

The effect of isoproterenol (IPR) on phosphorylation of acidic nuclear proteins was investigated by two-dimensional gel autoradiography. Mouse spleen cells stimulated to divide by the mitogen concanavalin A (Con A) were separated according to cell cycle stage by flow microfluorometric technique. Exposure of cells for 48 h to 4 micrograms IPR/ml culture medium produced no significant change in the proportion of S and G2 phase cells, while a cumulative dose of 8 micrograms IPR/ml caused a significant repression in DNA synthesis and a reduction in the number of nuclei in G2 + M phase. Four micrograms IPR/ml stimulated the greatest amount of G0 + G1 phosphorylation of nuclear protein. Several proteins from G0 + G1 and S nuclei incorporated 32P after Con A + IPR administration, and one protein from S phase nuclei revealed intensified labeling at the 8 micrograms cumulative IPR dose but not at the 4 micrograms dose. The isolated proteins (W, X, Y, and Z) were reassociated with homologous DNA, centrifuged in a sucrose gradient and shown to co-sediment with DNA. S phase nuclear protein X-S, which was found to be a mixture of proteins (X0 and X1), was the only exception. One component of X-S, X0 bound to DNA, while component X1 failed to bind. Chymotryptic and V8 protease digests of all isolated proteins were made and analyzed by autoradiography. Proteins X0 and X1, recovered from the sucrose gradient, possessed dissimilar fragment patterns. It is concluded that protein X-S is composed of two proteins (X0 and X1), one of which (X1) appears during S phase during the 8 micrograms IPR induced nuclear repression.