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Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase
Author(s) -
PerezReyes Edward,
Cooper Dermot M. F.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb01769.x
Subject(s) - cyclase , adenylate kinase , gtp' , inhibitory postsynaptic potential , component (thermodynamics) , chemistry , neuroscience , biochemistry , biology , enzyme , physics , thermodynamics
Functional interaction of the inhibitory GTP regulatory component (N i ) 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′‐(β‐γ‐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) > Gpp(NH)p > GTP. Inclusion of 0.1 m M 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 inhibitin of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional N i and stimulatory GTP regulatory component (N s ) activities. The sedimentation of functional N i 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 [ 32 P]NAD revealed that both the 39,000‐ and 41,000‐dalton substrates comigrated with the functional N i activity. These results demonstrate the feasibility of reconstituting N i inhibition with resolved catalytic activity directly in solution.