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 ([(3)H]
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) 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP), have similar subunit structures. The isolated
zona radiata
protein showed cooperativity of binding to [(3)H]17α,20β-DHP and PA labelling to [(3)H]
R5020. The association constant (Ka) was 2.0×10(7)M(-1) and maximum binding capacity (Nmax) 427 fmoles/mg
protein with MIS [(3)H]17α,20β-DHP.No evidence for nuclear binding of MIS [(3)H]17α,20β-DHP or PA labelling of [(3)H]
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 17α,20β-DHP receptor-like
protein from the
zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.