Binding of lipoprotein lipase to endothelial cells in culture.
Equilibrium-binding data of highly purified avian lipoprotein lipase to cultured bovine endothelial cells demonstrate the presence of a class of high affinity sites. Analysis of the binding function by weighted least squares technique yielded an association constant of K = 0.7 X 10(7) M-1 and a maximum binding capacity of 1.6 micrograms/1.9 X 10(6) cells. Lipoprotein lipase was monitored both by its catalytic activity and a sensitive radioimmunoassay which permitted the accurate measurement of nanogram quantities of enzyme protein. Specific activity of the bound enzyme was similar to that of the initial purified enzyme. Lipoprotein lipase binding to endothelial cells was inhibited 80% by preincubating cells in 0.1% trypsin for 3 min at 37 degrees C, 92% by 0.01% pronase, and 91% by 0.008% proteinase K. Heparin was most efficient in releasing lipoprotein lipase from endothelial cells. Fifty per cent of the enzyme appeared in the medium at a concentration of 3 micrograms/ml of heparin. At the same concentration of heparan sulfate, 20% of the enzyme was released. Hyaluronic acid and chondroitin sulfate were not effective in stimulating enzyme release. Preincubating endothelial cells with purified human platelet endoglucuronidase for 1 h at 37 degrees C led to a 90% reduction in lipoprotein lipase binding. Endoglucuronidase was purified 20,000-fold as compared to the initial platelet lysate by a 5-step purification method. The extent of inhibition of binding was shown to be dependent on concentration of endoglucuronidase in the preincubation medium. The specificity of platelet endoglucuronidase and the demonstration that the preparation utilized contained no detectable protease activity is further evidence that lipoprotein lipase is bound to endothelial cell heparan sulfate or heparan sulfate-like molecules.
D000818:Animals; D001011:Aorta / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology; D002417:Cattle; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D005966:Glucuronidase / Q000097:blood / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism*; D011863:Radioimmunoassay
D005966:Glucuronidase; D006493:Heparin; D006497:Heparan Sulfate; D008071:Lipoprotein Lipase