Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.
Cyclic GMP-dependent protein kinase was purified from foetal 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 cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-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 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.
D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D000242:Cyclic AMP; D006152:Cyclic GMP; D004573:Electrolytes / Q000494:pharmacology; D005318:Fetal Heart / Q000201:enzymology*; D006657:Histones; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009712:Nucleotides, Cyclic; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D004573:Electrolytes; D006657:Histones; D009712:Nucleotides, Cyclic; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP