Detection of a 58-Kilodalton High Density Lipoprotein-Binding Protein in the Membrane Fraction of Luteinized Rat Ovaries*

Abstract

The membrane fraction from the ovaries of pseudopregnant rats exhibits specific, high affinity binding of high density lipoproteins (HDL). Previous studies have indicated that HDL binding in this tissue is up-regulated by hCG and may be involved in supplying cholesterol as substrate for steroid hormone production. To characterize the HDL-binding activity, we solubilized the membrane proteins using 40 mM beta-octylglucoside and then separated them by electrophoresis on a 7% sodium dodecyl sulfate-polyacrylamide gel. The separated proteins were transferred to nitrocellulose sheets and subsequently incubated in the presence of [125I]apolipoprotein-E-free HDL. Autoradiography of the nitrocellulose revealed that the labeled HDL was bound to a single major band, with an apparent mol wt of 58,000 daltons. Neither reduction with beta-mercaptoethanol nor heat denaturation before separation on the gel affected the molecular size of the band, which indicates that it is probably a single polypeptide chain. The band was up-regulated in this tissue by in vivo treatment with 25 IU hCG in a time-dependent manner similar to the up-regulation of [125I]HDL-binding activity. In contrast to the binding of low density lipoprotein (LDL) to its receptor, the binding of HDL is independent of Ca+2. Incubation of the transferred proteins in the presence of [125I] Incubation of the transferred proteins in the presence of [125I]apolipoprotein-E-free HDL and either 5 mM CaCl2 or 15 mM EDTA had no effect on the appearance of the 58-kDa band. Furthermore, ligand blotting in the presence of a 100-fold excess of LDL did not affect the appearance of the band, whereas a 100-fold excess of apoliprotein-E-free HDL caused the disappearance of the band, indicating specificity for binding of HDL. Treatment of the sample with trypsin before electrophoresis also caused the band to disappear, revealing the protein nature of the band. These experiments indicate that the HDL receptor in luteinized rat ovaries is a 58,000-dalton protein.