Phospholipid-sensitive Ca2+-dependent protein kinase from bovine thyroid: characteristics and subcellular distribution of the enzyme and its substrates.
Ca2+-phosphatidylserine-dependent protein kinase activity was demonstrated in whole thyroid homogenates, cytosol, particulate, and membrane fractions. Although Ca2+-phospholipid-dependent protein kinase was difficult to detect in purified thyroid plasma membranes, an EGTA extract of such membranes had this activity. While phosphatidylinositol did not stimulate the enzyme, it enhanced the response to phosphatidylserine in the presence of 10 mmol/L free Ca2+. The enzyme was active at concentrations of free Ca2+ as low as 1 mumol/L but was inhibited at Ca2+ in excess of 500 mumol/L. Bovine thyroid contained endogenous substrates of 38,000 and 33,000 daltons for thyroid or purified spleen Ca2+-phospholipid-dependent protein kinase. The 38,000 dalton polypeptide was present in all the subcellular fractions while the 33,000 dalton substrate was present only in the whole homogenate and cytosolic fraction. The 38,000 dalton polypeptide, like the Ca2+-phospholipid-dependent protein kinase, was released from thyroid plasma membranes by EGTA. Phosphorylation of this substrate was rapid, highly sensitive to Ca2+, and inhibited by chlorpromazine (100 mumol/L) and trifluoperazine chlorpromazine (100 mumol/L) and trifluoperazine (100 mumol/L). Several substrates of a phospholipid-independent, Ca2+-dependent protein kinase with molecular weights of 51,000, 76,000, and 96,000 were also observed. This Ca2+-phospholipid-dependent protein phosphorylation system may be important in the membrane-associated functions of the thyroid.
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002746:Chlorpromazine / Q000494:pharmacology; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000201:enzymology* / Q000648:ultrastructure; D014268:Trifluoperazine / Q000494:pharmacology
D014268:Trifluoperazine; D004533:Egtazic Acid; D011493:Protein Kinase C; D002746:Chlorpromazine