Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.
The ovary uses the cholesterol from high-density lipoproteins (HDL) as a substrate source for steroid hormone production. It is not clear, however, how ovarian cells acquire the lipoprotein cholesterol. This study describes the characterization and isolation of a high-affinity-binding protein for apolipoprotein E-free HDL from the plasma-membrane fraction of bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment of 5'-nucleotidase activity. The binding of 125I-HDL to the plasma membranes was time-dependent, and there appeared to be a single high-affinity site with a Kd of 6.7 micrograms of HDL/ml of assay buffer. The binding was not affected by high concentrations of low-density lipoproteins or the Ca2+ chelator EDTA, nor by changes in pH in the range 6.5-9.0. The binding was affected by the salt concentration in the buffer, with a dose-dependent increase that reached a maximum at 150-250 mM-NaCl. Binding was increased in the presence of high concentrations of KCl and KBr, and most significantly increased by high concentrations of bivalent metal ions. Ligand-blot analysis under reducing conditions revealed that the binding protein was a single polypeptide of about 108 kDa that was associated with the plasma-membrane fraction. This HDL-binding protein was purified to homogeneity by solubilization with Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, and preparative SDS/PAGE. The purified binding protein is a single polypeptide of 108 kDa that retains high affinity and specificity for HDL as assayed by ligand blotting.
Ferreri
K
K
Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor 48109-0278.
Menon
K M
KM
D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002850:Chromatography, Gel; D003338:Corpus Luteum / Q000737:chemistry* / Q000648:ultrastructure; D005260:Female; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018110:Receptors, Lipoprotein
D002352:Carrier Proteins; D008075:Lipoproteins, HDL; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein