Effects of proteolytic enzymes and protease inhibitors on bovine thyroid adenylate cyclase activity.
Trypsin, chymotrypsin, and papain stimulate basal adenylate cyclase activity in bovine thyroid plasma membranes in a dose-related, albeit biphasic, fashion. Each of the proteases enhanced TSH-stimulated adenylate cyclase activity over basal activity. The proteases also enhanced GTP-, guanosine 5'-(beta, gamma-imidotriphosphate)-, prostaglandin E1-, and cholera toxin-stimulated adenylate cyclase to varying degrees. Fluoride-stimulated activity was enhanced by chymotrypsin and papain, but not by trypsin. When Mn++ was substituted for Mg++ in the adenylate cyclase assay, no stimulation by the proteases were observed. To see if endogenous membrane proteases are required for optimal thyroid adenylate cyclase response to TSH and other stimulators, studies were performed using the protease inhibitors tosylamide 2-phenylethyl-chloromethyl ketone (TPCK) and p-tosyl-L-arginine methyl ester (TAME), inhibitors of chymotrypsin and trypsin, respectively. TPCK (0.15 mM) had no effect on basal adenylate cyclase activity, but did inhibit TSH-, trypsin-, and chymotrypsin-stimulated activities by approximately 90%. Guanosine 5'-(beta, gamma-imido) triphosphate- as well as cholera toxin-stimulated activities were inhibited by approximately 50%, whereas prostaglandin E1- and fluoride-stimulated activities were inhibited by approximately 25%. TAME (6 mM) produced similar results, except that no effect on fluoride activity was seen, while basal activity was inhibited by approximately 20%. Thus, various serine proteases augment both basal and hormone-stimulated adenylate cyclase in bovine thyroid. Since both trypsin- and chymotrypsin-stimulated as well as TSH-induced enzyme activities were inhibited by TPCK and TAME, it would appear that augmentation of thyroid adenylate cyclase activity may, in part, result from stimulation of endogenous proteases.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002772:Cholera Toxin / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology*; D005459:Fluorides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D010206:Papain / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D013961:Thyroid Gland / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D002413:Cations, Divalent; D011480:Protease Inhibitors; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D013972:Thyrotropin; D002772:Cholera Toxin; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain; D000262:Adenylyl Cyclases; D005459:Fluorides