Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes.
This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.
D000262:Adenylyl Cyclases / Q000097:blood*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002772:Cholera Toxin / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000032:analysis; D014422:Turkeys
D006150:Guanine Nucleotides; D011943:Receptors, Adrenergic, beta; D009243:NAD; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide; D005459:Fluorides