Identification and purification of a bovine corpora luteal membrane glycoprotein with [3H]prostaglandin F2-alpha binding properties.
A bovine corpora luteal membrane glycoprotein which coelutes from multiple chromatographic procedures with bound tritiated prostaglandin F2a ([3H]PGF2 alpha) has been identified and purified to homogeneity. The properties of this molecule include: an apparent molecular mass by polyacrylamide gel electrophoresis (PAGE) of 135 kD; glycosylation which resists endoglycosidases D and H but is susceptible to cleavage by the exoglycosidase sialidase; binding of the molecule to Wheat Germ Agglutinin Sepharose but not to Concanavalin A Sepharose or Soybean Agglutinin Sepharose; migration on O'Farrell 2-D PAGE (pI 3-10) to the acidic side of the gel; binding to DEAE-Cellulose at pH 7.5 which can be displaced with NaCl at concentrations above approximately 100 mM; and, when solubilized with Triton X-100, binding to Phenyl-Sepharose or Octyl-Sepharose columns. Lastly, a rabbit polyclonal antibody against this [3H]PGF2 alpha binding protein has been made which allows both Western blotting of the 135 kD protein as well as immunohistochemical staining of ovarian tissue in a manner expected from previous binding studies. Problems associated with membrane solubilization of the receptor and receptor renaturation are discussed.
Orlicky
D J
DJ
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
Miller
G J
GJ
Evans
R M
RM
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003338:Corpus Luteum / Q000737:chemistry*; D015237:Dinoprost / Q000378:metabolism*; D005260:Female; D007106:Immune Sera; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011247:Pregnancy; D011817:Rabbits; D011982:Receptors, Prostaglandin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D007106:Immune Sera; D008562:Membrane Glycoproteins; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D015237:Dinoprost