Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

1. 1. Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. 2. 2. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nucleic, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol ³²P incorporated/hr/mg of enzyme. 3. 3. Protein kinase N1 comprised one polypeptide of M_r 35,000 which underwent phosphorylation in the presence of Mg²⁺ + ATP. Protein kinase N2 comprised two polypeptides M_r 40,000 and 30,000 of which only the M_r 30,000 polypeptide was autophosphorylated. 4. 4. Both enzymes were active towards casein, phosvitin, dephosphosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. 5. 5. Both enzymes were stimulated by 150-200 mM NaCl. MgCl₂ requirement varied with the protein substrate but was between 2-4 mM for both enzymes. 6. 6. With dephosphophosvitin as substrate, the apparent K_m for ATP for N1 protein kas 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent K_m was 0.01 mM for ATP, but 0.1 mM for GTP.