Abstract
Treatment of membranes with islet activating protein (IAP), a toxin from Bordetella pertussis, results in abolition of GTP-dependent, receptor-mediated inhibition of adenylate cyclase. This appears to result from IAP-catalyzed ADP-ribosylation of a 41,000-Da membrane-bound protein. A protein with 41,000- and 35,000-Da subunits has been purified from rabbit liver membranes as the predominant substrate for IAP. This protein has now been shown to be capable of regulating membrane-bound adenylate cyclase activity of human platelets under various conditions. The characteristics of the actions of the IAP substrate are as follows. 1) Purified 41,000/35,000-Da dimer is capable of restoring the inhibitory effects of guanine nucleotides and the alpha 2-adrenergic agonist, epinephrine, on the adenylate cyclase activity of IAP-treated membranes. 2) The subunits of the dimer dissociate in the presence of guanine nucleotide analogs or A1(3+), Mg2+, and F-. The 41,000-Da subunit has a high affinity binding site for guanine nucleotides. 3) The resolved 35,000-Da subunit of the dimer mimics guanine nucleotide- and epinephrine-induced inhibition of adenylate cyclase. 4) The resolved (unliganded) 41,000-Da subunit stimulates adenylate cyclase activity and relieves guanine nucleotide- +/- epinephrine-induced inhibition of the enzyme. In contrast, the GTP gamma S-bound form of the 41,000-Da subunit inhibits adenylate cyclase activity, although with lower apparent affinity than does the 35,000-Da subunit. 5) The 35,000-Da subunit increases the rate of deactivation of Gs, the stimulatory regulatory protein of adenylate cyclase. In contrast, the 41,000-Da subunit can interact with Gs and inhibit its deactivation. These data strongly suggest that the IAP substrate is another dimeric, guanine nucleotide-binding regulatory protein and that it is responsible for inhibitory modulation of adenylate cyclase activity.
Highlights
PROPERTIES AND FUNCTION OF THE PURIFIED PROTEIN*Reconin abolition of GTP-dependent, receptor-mediated in- stitution of adenylate cyclase activity ina genetic variant hibition of adenylate cyclase
From the $Department of Pharmacology, University of Texas Health Science Center aDt allas, Dallas, Texas 75235 and the
41,000-Da subunit inhibits adenylate cyclase activityt,o be described in thisreport substantiate thehypothesis that with lower apparent affinity than does the the substrate for IAP is the inhibitory guanine nucleotide
Summary
Reconin abolition of GTP-dependent, receptor-mediated in- stitution of adenylate cyclase activity ina genetic variant hibition of adenylate cyclase This appears to result (cyc-) of the mouse S49 lymphoma cell that has a deficiency from IAP-catalyzed ADP-ribosylationof a 41,000-Da in the hormone-stimulated adenylate cyclase complex serves membrane-bound protein. 2) The pertussis, resultsin the abolition of GTP-dependent and subunits of the dimer dissociatein the presence of receptor-mediated inhibition of adenylate cyclase [9,10,11].This guanine nucleotide analogsor A13+, Mgz+a,nd F-. 41,000-Da subunit inhibits adenylate cyclase activityt,o be described in thisreport substantiate thehypothesis that with lower apparent affinity than does the the substrate for IAP is the inhibitory guanine nucleotide-. The 41,000-Da subunit can interact wG.itahnd inhibit
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