Protein Kinases and Proteins Binding Adenosine 3': 5'-Monophosphate in Subcellular Fractions of Calf Ovaries. Effect of Trypsin and Protease Inhibitors on Protein Kinases

Abstract

Protein kinase activity was identified in the nuclear, microsomal, lysosomal-mitochondrial and soluble fractions isolated from calf ovaries. In all particulate fractions the protein kinase activity was only slightly stimulated by adenosine 3′: 5′-monophosphate (cAMP) as compared to a 5-fold stimulation of the soluble kinase activity. More than 80% of the enzyme activity measured in the presence of cAMP was found in the soluble fraction. Incubation with 0.2% Triton X-100 resulted in a 2–3-fold increase in the protein kinase activities in the lysosomal-mitochondrial and microsomal fractions. Triton X-100 solubilized 40–60% of the protein kinase and cAMP-binding activities in the three particulate fractions. Analysis of the Triton X-100 extracts and cytosol by Sephadex G-200 chromatography revealed multiple protein kinase and cAMP-binding peaks. The cytosol fraction contained only a 230000 molecular weight cAMP-dependent protein kinase (kinase-I). The nuclear extract prepared in the presence of the serine protease inhibitor, phenylmethanesulfonyl fluoride, exhibited the cAMP-dependent protein kinase with a molecular weight of 230000 (kinase-I). However in the absence of phenylmethanesulfonyl fluoride the proportion of this large enzyme was greatly diminished and there was a concomitant increase in the proportion of a smaller cAMP-dependent protein kinase of 88000 molecular weight (kinase-II). This conversion was accompanied by little loss of enzyme activity. A fragment of 88000 molecular weight retaining cAMP-dependent enzyme activity could also be produced by controlled trypsin digestion of the 230000 molecular weight holoenzyme, supporting the contention for a protease in the subcellular particles capable of splitting a susceptible bond in the large kinase-I. In the lysosomal-mitochondrial extract the major cAMP-dependent kinase eluted with a molecular weight of 88000, which did not alter in size in the presence of phenylmethanesulfonyl fluoride. The cAMP-dependent protein kinases in the particulate fractions appeared to be the same as, or cleavage products from, the soluble 230000 molecular weight holoenzyme. All three particulate fractions contained a cAMP-independent protein kinase of 40000 molecular weight (kinase-III). This enzyme can be classified as cAMP-insensitive as it was neither inhibited by the heat-stable inhibitor from rabbit muscle nor by the regulatory subunit of the soluble cAMP-dependent enzyme. Additional evidence that kinase-III is not the catalytic subunit of the cAMP-dependent enzymes comes from the different substrate specifities of the enzymes. A high affinity (Kd= 1.2 × 10−9 M) cAMP-binding protein with a molecular weight of 40000 was apparent in all particulate fractions. In the nuclear extract it represented the major cAMP-binding activity.