Mare Serum Gonadotropin
AND PHYSICOCHEMICAL, BIOLOGICAL,
form, November 28, 1978)
22, 1978, and in revised
and Om P. Bahl@
of Biological+ork 14260 Sciences, Division of Cell and Molecular Biology, State University of New York at
From the DeDartment Buffalo, Bufjalo, New
Procedures have been developed for the purification of pregnant mare serum gonadotropin (PMSG) and its a and jl subunits. The procedure for the hormone purification involves three steps of column chromatography on Sephadex G-100, DEAE-Sephadex A-50, andhydroxyapatite. The preparation of subunits involves the dissociation of PMSG with 10 M urea followed by their separation by chromatography on DEAE-Sephadex and Sephadex G-100. The hormone and subunit preparations were found homogeneous by electrophoresis in polyacrylamide gel with or without sodium dodecyl sulfate and by immunodiffusion. The hormone had an activity of 13,740 I.U./mg as determined byin viva and in vitro bioassays and receptor binding assays. The subunits did not show any significant activity by the receptor binding assay. The molecular weights of PMSG, PMSG-e, and PMSG-P, determined from Ferguson plots (Ferguson, K. A. (1964) Metabolism 13, 9851002) using glycoproteins as molecular weight markers, were 64,030,43,720 and 16,960, respectively. The amino acid and carbohydratecompositions of the hormone and the subunits have been determined. The carbohydrate content of the hormone was 41.7% and the (Y and jl subunits contained 20.6 and 45.6% carbohydrate, respectively (uncorrected for moisture content of protein). The carbohydrate moiety of the hormone is made up of L-fucose (0.6 to 0.9%), D-mannose (2.0 to 2.3%), Dgalactose (10.6 to 12%), N-acetylglucosamine (9.0 to10.5%), N-acetylgalactosamine (3.0 to 3.5%), and sialic acid (12.0 to 14.0%). The purified PMSG was found to be three times as active as ovine lutropin (LH, luteinizing hormone) (2.3 NIH-LH-SI units/mg) and 2/3 as active as ovine follitropin (FSH, follicle-stimulating hormone) (115.3 NIHFSH-SI units/mg). FSH activity was determined by the Steelman-Pohley assay (Steelman, S., and Pohley, F. (1953)Endocrinology 53, 604-616) and the LH activity was measured by ascorbic acid depletion assay. As determined by binding assay, the individual subunits upon recombination recovered 27.2% of the LH activity and 62.5% of the FSH activity. Immunologically, PMSGa and PMSG-j? cross-reacted with anti-PMSG as found by radioimmunoassay. While human chorionic gonadotropin (hCG) and human-luteinizing hormone(hLH) competed with ‘“‘I-PMSG in the PMSG receptor binding assay, they showed little or no cross-reactivity in the
* This work was supported by Research Grant HD-08766 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC.Section 1734 solely to indicate this fact. $ Present address, Department of Biochemistry, University of California, Riverside, Riverside, Calif. 92521. 5 To whom correspondence should be addressed.
radioimmunoassay, antibody binding
indicating that the sites are different from
receptor and each other.
Various glycoprotein hormones, such as luteinizing (LH), follicle-stimulating (FSH),thyroid-stimulating, and human chorionic gonadotropin (hCG), have been purified and characterized in detail (l-12). Little, however, is known about the detailed chemistry and properties of pregnant mare serum gonadotropin (PMSG), a glycoprotein hormone that possesses both FSH’ and LH activities. Various procedures utilizing commercial preparations have been described for the isolation of PMSG...