Hormonal modulation of guanyl nucleotide binding to rat luteal membranes.
Guanyl nucleotides are known to play a dual role in the activation of the adenylate cyclase system of the rat corpus luteum, being required for human choriogonadotropin (hCG) stimulation of the enzyme and modulating hCG binding to some hormone receptors. Current models of adenylate cyclase activation require that guanyl nucleotide binding be enhanced by hormones, and we have examined this binding in rat luteal membrane preparations known to contain guanyl nucleotide-modulated hCG receptors. [3H] Guanylyl-imidodiphosphate (GMPPnP), a nonhydrolyzable analog of guanosine triphosphate (GTP), was used to investigate binding to urea-washed, heavy rat luteal membranes. Binding was found to be linear, with respect to the amount of membranes added, in the range of 2-10 mg wet wt. tissue equivalents, and equilibrium was reached after a 30-min incubation at 30 degrees C. Analysis of equilibrium binding experiments gave a Ka of 1.2.10(7) +/- 0.9.10(7) M-1, with 460 +/- 430 fmol binding sites per mg tissue in the absence of hormone, Kinetic experiments showed an association rate constant of 2.6.10(5) +/- 0.5.10(5) M-1 min-1 and a dissociation rate constant of 1.8.10(-2) +/- 0.9.10(-2) min-1. In the presence of hCG, the Ka was unchanged; however, the number of binding sites increased by 50-120%. Competitive binding assays utilizing other nucleotides revealed that a hierarchy of GMPPnP = GTP greater than guanosine diphosphate (GDP) greater than inosine triphosphate (ITP) in displacing labeled GMPPnP. A similar hierarchy was also found for hCG-stimulated adenylate cyclase activity (GMPPnP = GTP greater than ITP) and for modulation of hCG binding (GMPPnP greater than GTP greater than ITP).(ABSTRACT TRUNCATED AT 250 WORDS)
McIlroy
P J
PJ
Department of Biology, Camden College of Arts and Sciences, Rutgers University, New Jersey 08102.
Taylor
C M
CM
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003338:Corpus Luteum / Q000201:enzymology / Q000378:metabolism*; D046508:Culture Techniques; D005260:Female; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051381:Rats; D011919:Rats, Inbred Strains; D011967:Receptors, Gonadotropin / Q000378:metabolism
D006063:Chorionic Gonadotropin; D006150:Guanine Nucleotides; D011967:Receptors, Gonadotropin; D007293:Inosine Triphosphate; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases