Receptor-mediated activation of detergent-solubilized guanylate cyclase.
Here for the first time we report the successful detergent-solubilization of the speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) receptor and the subsequent activation of guanylate cyclase in response to receptor occupation. Sea urchin sperm membranes treated with a solution containing 0.5% LubrolR PX and 0.5% EmulphogeneR in the presence of MgCl2 and NaF released both the speract receptor and guanylate cyclase activity into solution. The solubilized apparent receptor was not sedimented at 400,000 x g x 15 min and was not retained by glass microfiber filters. In the presence of 125I-GGG(Y2)speract and dissuccinimidyl suberate, a major radioactive band at about Mr = 77,000 and minor bands at Mr = 35,000 and 150,000 were cross-linked. Speract but not resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) competed in the cross-linking reaction. The amount of 125I-GGG(Y2)speract bound to solubilized receptor did not increase in a linear manner as a function of added protein but instead was concave upward. The addition of speract but not resact to the solubilized preparation resulted in the activation of the enzyme guanylate cyclase; the extent of stimulation was dependent on the amount of enzyme protein added and also was concave upward. Approximately 900 nM speract half-maximally activated guanylate cyclase. These data suggest that the speract receptor and guanylate cyclase are closely apposed, even in detergent, or that they are the same molecule.
Bentley
J K
JK
Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
Khatra
A S
AS
Garbers
D L
DL
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000502:physiology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D008297:Male; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000502:physiology; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000187:drug effects* / Q000201:enzymology; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D006023:Glycoproteins; D011956:Receptors, Cell Surface; D013501:Surface-Active Agents; C035027:egg surface sperm receptor; D006162:Guanylate Cyclase