Interaction of the inhibitory GTP regulatory component with soluble cerebral cortical adenylate cyclase.
Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Perez-Reyes
E
E
Cooper
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D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001088:Arabinonucleotides / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000201:enzymology*; D002850:Chromatography, Gel; D004533:Egtazic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D013873:Thionucleotides / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D001088:Arabinonucleotides; D013873:Thionucleotides; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; C022206:9-beta-D-arabinofuranosylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide