Kinetics of product inhibition and mechanisms of lipoprotein lipase activation by apolipoprotein C-II.
The kinetics of product inhibition of bovine milk lipoprotein lipase (LPL) were studied in a system of emulsified trioleoylglycerol (TG) at different fixed initial concentrations of oleic acid [( OA]0) without a fatty acid (FA) acceptor. In the absence of apolipoprotein C-II (C-II), the apparent Vmax and the nH(TG) (the slope of the corresponding Hill plot for TG) of 1.82 decreased by about 52% and [TG]0.5 increased 13-fold by raising the [OA]0 to 0.3 mM. At low [OA]0, product inhibition was competitive with respect to TG: the nH(OA) averaged 1.1, and [OA]0.5 was increased about 2-fold by TG. At the higher [OA]0, nH(OA) was 3.5, and TG had no effect on [OA]0.5. In the presence of 3 micrograms/mL C-II, the apparent Vmax was 4.3-7.1-fold higher than in its absence, and the nH(TG) was 2.45. Both parameters decreased by only 20-25%, and [TG]0.5 increased only 3-fold at an [OA]0 of 0.3 mM. Conversely, nH(OA) decreased by 35% and [OA]0.5 increased 6-fold by increasing TG concentrations. Similar kinetics were observed with very low density lipoproteins (VLDL). At saturating TG and varying C-II concentrations, nH(C-II) was 1.78, and product inhibition was found to be competitive with respect to C-II. At the [OA]0 employed, the FA had no effect on enzyme binding to TG emulsions, and there was no evidence that LPL catalyzes the reverse reaction. It is concluded that (a) the LPL kinetics are those of a multisite enzyme that probably has three high-affinity binding sites for TG, two for C-II, and four for OA.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000097:blood / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D005246:Feedback; D005260:Female; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors*; D008079:Lipoproteins, VLDL / Q000097:blood / Q000494:pharmacology; D008892:Milk / Q000201:enzymology; D014304:Triolein / Q000494:pharmacology
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D008079:Lipoproteins, VLDL; D014304:Triolein; D008071:Lipoprotein Lipase