Stimulatory modulator of cyclic GMP-dependent protein kinase was purified to homogeneity from heart extracts. The preparation appeared as a single protein band in the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, with a minimal M(r) of 34,000, which agreed with the value calculated from its amino acid composition. The modulator was an acidic protein, as indicated by an isoelectric point of about pH 4.0 and a high content of acidic amino acids. It had a Stokes radius of 62 Å and a frictional ratio (f/f 0) of 2.27, indicating that the factor is a highly elongated or asymmetric protein. The modulator exhibited multiple bands in analytical polyacrylamide gel electrophoresis, with each band found to be active. It had a sedimentation coefficient of 2.7 S and a calculated M(r) of 69,000. The 'reassociated' modulator, obtained by dialysis of the SDS-treated factor, was nearly as active as and exhibited the same characteristics of the 'native' modulator mentioned above. The modulator augmented the cyclic GMP-dependent phosphorylation of histones, but had little or no effect on phosphorylation of other proteins such as protamine, myelin basic protein, a synthetic heptapeptide (Kemptide), troponin, and glycogen synthetase. A maximal phosphorylation of histones, as a result of a high activation by cyclic GMP elicited by the modulator, was seen at a Mg 2+ concentration of about 10 to 20 mM. At higher Mg 2+ concentrations (50 to 100 mM), however, the depressed phosphorylating activity became more directly stimulatable by cyclic GMP, and furthermore, this activation was not augmented by the modulator. It had no effect on the binding of radioactive cyclic GMP and cyclic AMP to their respective receptor protein kinases. The modulator interacted with histones, but not with the putative catalytic subunit of cyclic GMP-dependent protein kinase, nor the catalytic subunit of the cyclic AMP-dependent enzyme. It is suggested that the modulator augmented phosphorylation of histones, or probably histone-like proteins, at least in part by interacting with the proteins, thus rendering them more effective substrates for the enzyme.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1978|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology