Doubly radioiodinated luteinizing hormone: preparation and characterization.
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.
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D002417:Cattle; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D007985:Leydig Cells / Q000187:drug effects; D007986:Luteinizing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface / Q000378:metabolism; D011974:Receptors, LH
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011974:Receptors, LH; C031857:luteinizing hormone, diiodo; D007986:Luteinizing Hormone