Demonstration of putative membrane and cytosol steroid receptors for 17α, 20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020) (original) (raw)

Abstract

Cytosol from brook trout ovarian follicles (stages 1–3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([3H]R5020). The covalently bound cytosol protein had a relative mass of 501,000 Mr following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6–7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 Mr. The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17_α_,20_β_-dihydroxy-4-pregnen-3-one (17_α_,20_β_-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [3H]17_α_,20_β_-DHP and PA labelling to [3H]R5020. The association constant (Ka) was 2.0×107M−1 and maximum binding capacity (Nmax) 427 fmoles/mg protein with MIS [3H]17_α_,20_β_-DHP.

No evidence for nuclear binding of MIS [3H]17_α_,20_β_-DHP or PA labelling of [3H]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17_α_,20_β_-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.

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1. A. Maneckjee
   Present address: Health and Welfare Canada, Biotechnology Div., Banting Building, Tunney's Pasture, K1A 0L2, Ottawa, Ontario, Canada

Authors and Affiliations

1. Ocean Sciences Centre/Marine Sciences Research Laboratory, Memorial University of Newfoundland, A1C 5S7, St. John's, Newfoundland, Canada
   A. Maneckjee & D. R. Idler
2. Department of Biology, University of Regina, S4S 0A2, Regina, Saskatchewan, Canada
   M. Weisbart

Authors

1. A. Maneckjee
2. D. R. Idler
3. M. Weisbart

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Maneckjee, A., Idler, D.R. & Weisbart, M. Demonstration of putative membrane and cytosol steroid receptors for 17_α_, 20_β_-dihydroxy-4-pregnen-3-one in brook trout_Salvelinus fontinalis_ oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020).Fish Physiol Biochem 9, 123–135 (1991). https://doi.org/10.1007/BF02265128

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• Accepted: 21 August 1990
• Issue date: April 1991
• DOI: https://doi.org/10.1007/BF02265128

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