Effect of the apolipoprotein [formula omitted] ratio on the capacity of purified milk lipoprotein lipase to hydrolyse triglycerides in monolayer vesicles

Abstract

The effect of the apolipoprotein C-II C-III 1 ratio on the capacity of purified bovine milk lipoprotein lipase to hydrolyse triglycerides was measured in a controlled model of pyrene-labeled nonanoyltriglycerides (1–2 ditetradecyl 3-pyrene nonanoyl glyceride) monolayer vesicles. Monolayer was composed of triglycerides, a non-hydrolysable phospholipid ether and cholesterol, a model system where the quality of the interface can be controlled. LPL released fatty acids from pyrene-triglycerides which were transferred from the lipoprotein structure to albumin. This transfer induces a decrease in the excimer production and in the excimer fluorescence intensity. Apolipoprotein C-II and C-III 0 and C-III 1 were purified from apolipoprotein VLDL. The 2 fragments, C-III 1 A (peptide 1–40) and C-III 1 B (peptide 41–79), were obtained after thrombin cleavage. Apolipoproteins C-III 0 and C-IIII 1 had a similar inhibitory effect on LPL. Inhibition with apo C-III 0 or apo C-III 1 was 85% of full LPL activity without inhibitor: Apo C-III 1 B inhibited 62% of basal activity. It was 27% less effective than apo C-III 1. Fragment C-III 1 A did not inhibit LPL. The effect of change in both apo C-II (0–0.6 μM) and apo C-III 1 (0–1.0 μM) on triglyceride hydrolysis shows the importance of the apo C-II C-III 1 ratio for the release of free fatty acids from triglycerides by LPL. The activating effect of apo C-II in the absence of the apo C-III inhibitor was maximal at 0.06 μM. No further activation was obtained between 0.06 and 0.30 μM. Higher concentrations decreased LPL activity. Apo C-III 1 (0.1 μM) decreased the maximum activation by apo C-II from 0.0196 to 0.063 nmol/min/nmol LPL. Higher concentrations of apo C-III 1 (0.1–0.5 μM) required higher apo C-II concentrations (0.30 μM instead of 0.06 μM) for maximal activation than when apo C-III 1 was absent. The activity of the enzyme without apo C-II was decreased by 65% by 0.12 μM apo C-III 1. Increasing the apo C-II apo C-III 1 ratio from 0.1 to 1, increased the activation of the enzyme by a given apo C-II concentration. Moreover, for a given apo C-II C-III 1 ratio, the LPL activation increased with the apo C-II concentration (between 0 and 0.010 μM), until a plateau was reached. This is important, as the change in the C-II C-III 1 ratio is not the only factor affecting LPL activity, and inhibition by apo C-III 1 also depends on the overall quantity of apolipoproteins. Extrapolation of these results suggests that hyperlipoproteinemia seems to be more likely due to overproduction of VLDL, than to a decrease in lipoprotein lipase activity.