Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit

Abstract

Cyclic[c]GMP-dependent protein kinase was purified from fetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cAMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cGMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cAMP-dependent protein kinase could not stimulate nor inhibit the cGMP target enzyme. The enzyme had Ka [association constant] values of 0.013, 0.033 and 3.0 .mu.M for 8-bromo cGMP, cGMP and cAMP, respectively. The cGMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM, respectively. The pH optimum for the enzyme activity ranged from 6-9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cAMP-dependent class of protein kinase. The holoenzyme (apparent MW 150,000) of the myocardial cGMP-dependent protein kinase was dissociated into a cGMP-independent catalytic subunit (apparent MW 60,000) by cGMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cGMP.