The reaction of tetranitromethane with human chorionic gonadotropin

Carlsen, Robert B.; Bahl, Om P.

doi:10.1016/0003-9861(76)90501-4

Cited by 20 publications

(4 citation statements)

References 29 publications

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“…Most studies of the role of the subunits have been based on the reduced ability of chemically modified gonadotropin analogs to bind to the receptor, initiate a biological response, or both (1,6,7). Recently, Ji and Ji (8) have made a photoaffinity label of hCG, which they linked to proteins on the surface of granulosa cells and which permitted them to radiolabel the receptor.…”

mentioning

confidence: 99%

Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Moyle¹,

Ehrlich²,

Canfield³

1982

Proc. Natl. Acad. Sci. U.S.A.

114

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Monoclonal antibodies were prepared against the a and f8 subunits of human chorionic gonadotropin (hCG). Although all were selected on the basis of their ability to bind the intact hormone, each also bound one of the two subunits but not both. Using a solid phase double antibody system to measure the relative binding to sites on the surface of hCG, we observed that four ofthe five antibodies bound to different sites on the molecule. This information was correlated with the ability of each antibody to inhibit the biological activity of hCG. Of the five antibodies tested for their ability to inhibit hCG-induced stimulation of rat testes steroidogenesis in vitro, two proved to be potent inhibitors, whereas the other three had almost no effect. This inhibition of steroidogenesis was highly correlated with the ability of the antibodies to inhibit hCG binding to testes homogenates. Thus, we have begun to derive a scheme that describes the relative binding positions of individual monoclonal antibodies and receptor on hCG. The purified monoclonal antibodies were iodinated and employed to evaluate which antigenic sites on hCG remained free in hCG-receptor complexes. The data indicated that portions of the g subunit in hCG-receptor complexes were buried (i.e., failed to bind radiolabeled antibody), whereas other portions remained exposed (i.e., they bound radiolabeled antibody). Those antibodies that interacted with portions of hCG that became inaccessible in the receptor complex also blocked the biological actions of hCG, whereas those that interacted with exposed sites had little or no effect on activity. Although we did not find antibodies to the a subunit that would bind to the hormone-receptor complex, we found that one of the two antibodies specific to a subunit epitopes blocked the actions of the hormone. Both antigenic determinants on the at subunit appeared to be lost after the hCG-receptor complex had formed. These studies suggest that each hCG subunit participates in the hormone-receptor complex and that portions of the ,B subunit project from the surface of the receptor.Human chorionic gonadotropin (hCG) is a glycoprotein hormone, composed of a and ,B subunits, which interacts with gonadal receptors to stimulate steroid synthesis (1). The isolated subunits exhibit less than 0.1% of the activity of the intact hormone and, within the limits of highest purity available, are thought to lack biological activity (1-3). As the subunits recombine, they reacquire biological activity (4), and spectral studies indicate that the conformation of the subunits is altered when they are recombined (5), suggesting that unique structural properties of the intact hormone may be needed for activity. Because neither subunit is active alone, the relative roles of the subunits in the actions of the hormone have been difficult to assess. Conceivably, only one of the subunits comes into direct contact with the receptor.Although the binding of hCG and related gonadotropins to Leydig cells has been studied at length (1), data con...

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mentioning

confidence: 99%

Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Moyle¹,

Ehrlich²,

Canfield³

1982

Proc. Natl. Acad. Sci. U.S.A.

114

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“…There is no tyrosine equivalent to bovine, ovine, and porcine LHa position 21 in hCG. Nitration studies have shown that a Tyr 92 and Tyr 93 can be modified in intact hCG, that Tyr 69 and Tyr 41 become available in free a, and that modification of position 92, 93, or 69 does not impair reassociation (27,28). The relative reactivities of hCGa tyrosines to iodination have not been studied.…”

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confidence: 96%

Doubly Radioiodinated Luteinizing Hormone: Preparation and Characterization*

Sharp

Pierce

1981

Endocrinology

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Bovine [131I]Iodo-alpha LH-[125I]iodo-beta LH (**LH) has been prepared and shown to be physically and biologically equivalent to unmodified hormone. The beta-subunit was modified with 125I, purified by adsorption to Concanavalin A-Sepharose and elution with methylmannoside, added to alpha-subunit, and allowed to reassociate to intact hormone. Iodination with 131I was then carried out in the reassociation mixture and **LH was isolated by gel filtration. Both gel electrophoresis and rechromatography on Sephadex G-100 showed that both radiolabels comigrated with unmodified hormone. Sodium dodecyl sulfate gel electrophoresis showed that 131I was found in the alpha-subunit and 125I in the beta-subunit; this result is in agreement with studies by others which show that the tyrosines of the beta-subunit are nonreactive in intact hormone. In receptor-binding assays, both radiolabels were specifically displaced in a similar fashion by LH. Scatchard analysis showed high affinity binding (Ka approximately equal to 1.5 X 10(10) M-1) for both labels. Comparison of receptor-binding activity with steroidogenic activity showed that iodinated hormone molecules not only bound to receptor but also stimulated testosterone production. The demonstration that full biological activity is retained with iodination in both subunits shows that such doubly labeled LH can be used to monitor the disposition of both subunits simultaneously during interaction of the hormone with target cells.

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“…Since neither subunit alone can effectively bind to the receptor with high affinity or exert biological effect (2,3), the direct interaction of the subunits in the hormone-receptor complex has been difficult to assess. Investigators have approached this problem by chemical modification of either a or /3 subunit in the intact hCG, followed by examining the binding and biological activity of the modified hCG (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). With this approach, it has been reported that both subunits of hCG interact with receptor (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

mentioning

confidence: 99%

“…Investigators have approached this problem by chemical modification of either a or /3 subunit in the intact hCG, followed by examining the binding and biological activity of the modified hCG (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). With this approach, it has been reported that both subunits of hCG interact with receptor (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, information regarding the spatial relationship of the subunits of hCG in the hormone-receptor complex is still obscure.…”

mentioning

confidence: 99%

Spatial relationships of the human chorionic gonadotropin (hCG) subunits in the assembly of the hCG-receptor complex in the luteinized rat ovary.

Hwang¹,

Menon²

1984

Proc. Natl. Acad. Sci. U.S.A.

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In an attempt to examine the spatial relationships of the human chorionic gonadotropin (hCG) subunits in the assembly of the hCG-receptor complex, the recombined '251-labeled hCG, with label in either the a subunit or the 18 subunit, was cross-linked to the luteinizing hormone (LH)/ hCG receptor. The efficacy of the cross-linking of the 125I-a subunit or the 125I-f subunit of hCG to the LH/hCG receptor was then examined. The autoradiographic profile of 25I1 hCG-receptor complex containing the label in the a subunit of hCG showed that the a subunit can cross-link with all four subunits of the LH/hCG receptor. However, only one faint labeled band, corresponding to Mr = 68,000, was detected when the 125I-hCG-receptor complex with label in the j8 subunit was subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions. When the electrophoresis was performed under nonreducing conditions, the Mr 68,000 band disappeared concomitantly with the accumulation of radioactivity in the high molecular weight region.These results indicated that the (?subunit of hCG, unlike the a subunit, can cross-link only weakly with the smallest subunit of the LH/hCG receptor. A comparison of the differential effectiveness of the cropsslinking of 1251-a subunit with 125-4I-subunit of hCG to the LH/hCG receptor suggests that both a and (3 subunits of hCG are intimately associated with the receptor, but the bulk (f the 13 subunit of hCG is buried in between the receptor and the a subunit of hCG. On the basis of our data, a model for the spatial arrangement of hCG subunits in the hCG-receptor complex is proposed.Human chorionic goqadotropin (hCG), a glycoprotein hormone, is composed of two dissimilar noncovalently bound subunits designated as a (Mr = 16,000) and A3 (Mr = 22,000) (1). The a subunit is common to all glycoprotein hormones and the /3 subunit coqfers the biological activity of the hormone. However, binding to the receptor occurs only when the hormone is in the a,8 form. Since neither subunit alone can effectively bind to the receptor with high affinity or exert biological effect (2, 3), the direct interaction of the subunits in the hormone-receptor complex has been difficult to assess. Investigators have approached this problem by chemical modification of either a or /3 subunit in the intact hCG, followed by examining the binding and biological activity of the modified hCG (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). With this approach, it has been reported that both subunits of hCG interact with receptor (4-14). However, information regarding the spatial relationship of the subunits of hCG in the hormone-receptor complex is still obscure. In this communication, we report the results obtained by using a chemical cross-linking approach to probe the spatial relationships of the subunits of hCG in its complex with receptor. Using this probe, we have compared the effectiveness of the cross-linking of the a subunit of hCG to the luteinizing hormone (LH)/hCG receptor with that of the P subunit. On...

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The reaction of tetranitromethane with human chorionic gonadotropin

Cited by 20 publications

References 29 publications

Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Doubly Radioiodinated Luteinizing Hormone: Preparation and Characterization*

Spatial relationships of the human chorionic gonadotropin (hCG) subunits in the assembly of the hCG-receptor complex in the luteinized rat ovary.

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