Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria

Abstract

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly( l-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent M r ~ 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain α-keto acid dehydrogenase (<3%), and it was essentially inactive (<0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 μg/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca 2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly( l-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent M r 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 m NaCl. Two subunits, with apparent M r's 36,000 (α) and 28,000 (β) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its β-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 μg/ml of heparin. Spermine (1.0 m m) stimulated activity of the purified kinase two- to threefold at 1.5 m m Mg 2+. Half-maximal stimulation occurred at 0.1 m m spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (<1%) toward pyruvate dehydrogenase and branched-chain α-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.