Covalent labelling of the lutropin binding site. Evidence for a single Mr 90000 sialoglycopolypeptide.
Membrane-associated sialoglycopolypeptides of rat ovaries were oxidized with NaIO4, reduced with NaB3H4 and solubilized with Triton X-100. The solubilized proteins carrying the 3H label were subjected to affinity chromatography on human choriogonadotropin coupled to agarose. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate followed by fluorography revealed a single component of apparent Mr 90000. This component was abolished when ovaries saturated with choriogonadotropin were used as starting material. The above result is identical to that obtained previously by conventional detection methods [ Metsikk ö & Rajaniemi (1982) Biochem. J. 208, 309-316] and indicates that the 3H-labelled lutropin/choriogonadotropin sialoglycopolypeptide was observed. The affinity-purified 3H-labelled protein co-eluted with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovarian particles, showed a Stokes' radius of 6.2 nm and sedimented as a single band with a sedimentation coefficient of 5.1 S. The sedimentation coefficient of this 3H-labelled protein was not significantly altered when boiled in 1% sodium dodecyl sulphate, indicating that non-covalently associated subunits were not present. The 3H-labelled protein cosedimented with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovary. When 125I-choriogonadotropin-receptor complex was covalently crosslinked with glutaraldehyde, an Mr 130000 component was produced as detected by sodium dodecyl sulphate gel electrophoresis. This component was extracted from the polyacrylamide gel and subjected to sucrose-density-gradient centrifugation in 0.1% Triton X-100. A single band sedimenting at the position of the 125I-choriogonadotropin-receptor complex solubilized from a prelabelled ovary was observed, exhibiting a sedimentation coefficient of 6.5S. These data suggest that the lutropin-binding site is a single sialoglycopolypeptide of Mr 90000, which binds one molecule of hormone resulting in an apparent Mr 130000 complex. The large Stokes' radius (6.2 nm) of the binding site is accounted for by bound detergent.
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D007986:Luteinizing Hormone / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010053:Ovary / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012795:Sialoglycoproteins / Q000032:analysis*
D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D012795:Sialoglycoproteins; D007986:Luteinizing Hormone