Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.

摘要: 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 lipoprotein cholesterol. This study describes characterization and isolation of high-affinity-binding protein apolipoprotein E-free HDL plasma-membrane fraction bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment 5'-nucleotidase activity. binding 125I-HDL to plasma was time-dependent, there appeared be single high-affinity site Kd 6.7 micrograms HDL/ml assay buffer. affected high concentrations low-density or Ca2+ chelator EDTA, nor changes in pH range 6.5-9.0. salt concentration buffer, dose-dependent increase that reached maximum at 150-250 mM-NaCl. Binding increased presence KCl KBr, most significantly bivalent metal ions. Ligand-blot analysis under reducing conditions revealed polypeptide about 108 kDa associated fraction. HDL-binding purified homogeneity solubilization Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, preparative SDS/PAGE. retains affinity specificity assayed ligand blotting.