Calcium-activated, calmodulin-dependent protein kinase activity in bovine thyroid cytosol.
Bovine thyroid 100,000 X g supernatant contained calcium-activated, calmodulin-dependent protein kinase (PK-CaM) activity. The PK-CaM was partially purified using ion-exchange chromatography and characterized. PK-CaM, using casein as exogenous substrate, was not stimulated by Ca2+(0-500 microM) or calmodulin (1-10 micrograms) by themselves, but was stimulated by the combination of the two by 100%. The activation of the enzyme by Ca2+ and calmodulin was dose-dependent with maximal stimulation evident at 1 microM free-Ca2+ and 3 micrograms calmodulin. Both chlorpromazine and trifluoperazine inhibited the thyroid enzyme in a dose-related manner. The molecular weight (MW) of the PK-CaM, based on gel filtration, was approximately 500,000. PK-CaM could also be demonstrated using endogenous thyroid cytosol proteins as substrate. Separation of these 32P-labelled proteins by SDS-PAGE and subsequent autoradiography revealed that one major protein of approximately 56,000 MW was phosphorylated by PK-CaM. In some experiments, a second, less-intense protein band of approximately 64,000 MW was also phosphorylated. Evidence is presented, suggesting that these two protein bands may result from the autophosphorylation of the PK-CaM holoenzyme. These results offer a molecular mechanism, in addition to protein kinase C, by which Ca2+ effects may be mediated in thyroid.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000201:enzymology*
D002147:Calmodulin; D011506:Proteins; D011494:Protein Kinases; D002118:Calcium