Effects of proteinase inhibitors on adenylate cyclase

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Abstract
The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N.alpha.-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low MW proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulfonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cylic[c]AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5''-[.beta..gamma.-imido]triphosphate or NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had 2 effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (.ltoreq. 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum 1st-order inhibitory rate constants of 0.05-0.08/min. At higher concentrations the irreversible effect persisted, but there was a marked decrease in the cyclase initial velocity to 25-50% of that of control values. N.alpha.-tosylarginine methyl ester had similar effects; at low concentrations (.ltoreq. 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (> 3 mM) N.alpha.-tosylarginine methyl ester inhibited NaF- and guanosine 5''-[.beta..gamma.-imido]triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in 2 ways, as for human-choriogonadotropin-stimulated cyclase, but required 10- to 20-fold higher concentrations. The low concentration irreversible effect can be explained by a continual inactive .dblarw. active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high concentration effect is a direct one on the active catalytic moiety of the enzyme.