Evidence that partially desialylated variants of human chorionic gonadotropin (hCG) are the factors in crude hCG that inhibit the response to thyrotropin in human thyroid membranes.
Previous studies have revealed that pure unmodified hCG (hCGp) has little potency to inhibit the binding of bovine TSH (bTSH) to human thyroid membranes and to either stimulate adenylate cyclase or inhibit TSH-stimulated adenylate cyclase therein. On the other hand, preparations of crude hCG (hCGc) as well as enzymatically desialylated hCGp (asialo-hCGp) are relatively potent inhibitors of bTSH binding (TBI activity) and bTSH-stimulated adenylate cyclase activity in human thyroid membrane preparations. In the present studies we have sought to identify and characterize the inhibitory moieties in crude hCG that are responsible for these inhibitory activities and to elucidate the properties of their interaction with the TSH receptor in human thyroid membranes. Preparations of hCGc were processed by DEAE-52 chromatography; this separated components of interest, which were not adsorbed, from intact hCGp, which was adsorbed to the column. The former were then subjected to gel filtration on Sephadex G-100, and the earliest eluting fractions, which proved to have the greatest TBI activity, were pooled, rechromatographed, and designated as variant hCG (hCGv). Three different preparations of hCGv were studied. All displayed a lower sialic acid content, by about half, than that in hCGp. Though their potencies varied somewhat, all had significant TBI activity, which was less than that of asialo-hCGp, but more than that of hCGc. Saturation studies revealed that the TBI activities of hCGv and asialo-hCGp were due to a competitive inhibition of bTSH binding at both the high and low affinity bTSH-binding sites, whereas the inhibitory activity of hCGc was exerted primarily at the low affinity binding site. Preparations of hCGv were also capable of inhibiting the adenylate cyclase response to TSH in human thyroid membranes, and Lineweaver-Burk analysis revealed this inhibition to be competitive in nature. As with its TBI activity, the potency of hCGv to inhibit the adenylate cyclase response was intermediate between that of asialo-hCGp and hCGc. Among the three batches of hCGv, their inhibitory effects on bTSH binding and adenylate cyclase activation appeared to vary inversely with with their sialic acid content. Enzymatic desialylation of hCGv increased its potency to that of asialo-hCGp. Several lines of evidence, as follows, indicate that the moieties that comprise hCGv are modified forms of hCGp itself. 1) On a unit weight basis, hCGv was at least as potent as hCGp in its ability to inhibit the binding of [125I]hCG to rat testicular membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
Hoermann
R
R
Charles A. Dana Research Institute, Harvard Thorndike Laboratory, Boston, Massachusetts.
Amir
S M
SM
Ingbar
S H
SH
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001212:Asialoglycoproteins; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D013737:Testis / Q000378:metabolism; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology*
D001212:Asialoglycoproteins; D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D012794:Sialic Acids; C019263:asialo-human chorionic gonadotropin; D013972:Thyrotropin; D000262:Adenylyl Cyclases