Apolipoprotein-E-binding proteins of rat liver endothelial cells

Abstract

In an attempt to characterise the apolipoprotein-E-binding proteins of rat liver endothelial cells, we prepared membranes from monolayer cultures of liver endothelial cells as an enriched source of membrane receptors. The membranes could specifically bind iodinated very-low-density lipoproteins (VLDL) and the binding could be inhibited effectively by unlabelled VLDL and high-density lipoproteins, but only moderately by low-density lipoproteins. To identify the binding proteins, we performed immunoprecipitation studies of solubilised iodinated liver endothelial cells and cell membranes, respectively, using purified apolipoprotein E and monospecific polyclonal IgG directed towards this apolipoprotein. The antibodies together with the bound apolipoprotein E and iodinated liver endothelial cell proteins were harvested with staphylococcal protein A-Sepharose. The immunoprecipitates were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and after autoradiography of the dried gel, the Mr of the liver endothelial cell proteins bound to apolipoprotein E could be determined. Two protein bands with molecular masses of 55-60 and 110, and a weak band of 170 kDa could be detected from intact cells. These proteins were specifically precipitated only in the presence of divalent cations, and might represent cell-surface receptors for apolipoprotein-E-containing lipoproteins. Additional bands were seen when cell membranes were used, the most prominent ones having molecular masses of 32 and 35 kDa. These proteins could be of intracellular origin, or they may be degradation products of the other apolipoprotein-E-binding proteins.