title: The comparative kinetics of soluble and heparin-Sepharose-immobilized bovine lipoprotein lipase.
abstract: While lipoprotein lipase (LPL) acts in vivo as an immobilized enzyme, its kinetics are commonly studied with soluble LPL (S-LPL). Hence kinetic parameters of S-LPL and heparin-Sepharose-immobilized LPL (B-LPL) were compared. A modified purification procedure for bovine milk, LPL gave a 56% yield of S-LPL, purified 7250-fold, and a specific activity of 27,000 mumol fatty acid/mg LPL/h when assayed with triolein (TG) emulsions in the presence of serum. The purified LPL also showed low but detectable esterase activity with p-nitrophenylacetate and p-nitrophenylbutyrate as substrates. Apolipoprotein C-II (C-II) had no effect on the esterase activity of LPL. Dixon plots of experiments with S-LPL indicated that heparin is a competitive inhibitor against both C-II and TG, and that the binding of either C-II or heparin to the enzyme is a mutually exclusive event. Similarly, the binding of TG and heparin to the enzyme is mutually exclusive. From the Dixon plots, the dissociation constant Ki for the LPL:heparin binary complex was determined to be 5.0 X 10(-8) M. In contrast to the heparin inhibitory effect on LPL activity against triolein, heparin had no effect on the esterase activity of LPL against p-nitrophenylacetate or p-nitrophenylbutyrate. Comparative studies with B-LPL and S-LPL, using triolein as substrate and apolipoprotein C-II or serum as activator, indicated that S-LPL has a higher apparent Km and lower apparent Vmax than B-LPL. It is concluded that most of the LPL bound to heparin-Sepharose is probably inaccessible to substrate, hence a low Vmax. However, Km (C-II) and Km (TG) were higher for B-LPL due to the competitive inhibitory effect of heparin on LPL. Consistent with these kinetic analyses and with the use of human very low density lipoproteins (VLDL) as substrate, S-LPL, even in the presence of heparin, was found to have an apparent rate of lipolysis of VLDL approximately ninefold greater than B-LPL.
mesh_terms: D000818:Animals; D053304:Apolipoprotein C-II; D001053:Apolipoproteins / Q000097:blood; D001056:Apolipoproteins C; D002417:Cattle; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005260:Female; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000302:isolation & purification / Q000378:metabolism*; D008892:Milk / Q000201:enzymology; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D053304:Apolipoprotein C-II; D001053:Apolipoproteins; D001056:Apolipoproteins C; D004800:Enzymes, Immobilized; C037405:heparin-sepharose; D006493:Heparin; D012685:Sepharose; D008071:Lipoprotein Lipase