Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding

Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies o f apoC-II and overexpression of apoC-II in transgenic mice are both associa ted with severe hyperlipidemia, indicating a complex role for apoC-II in th e regulation of blood lipid levels. ApoC-II exerts no effect on the activit y of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide correspon ding to amino acid residues 39-62 of mature human apoC-II. This peptide doe s not bind to model lipid surfaces but retains the ability to activate LpL, Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of t he affinity of the peptide for LpL using fluorescence anisotropy measuremen ts. The dissociation constant describing this interaction was 0.23 mu M, an d was unchanged when LpL was lipid-bound. Competitive binding studies showe d that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We sugges t that while the binding of apoC-II to the lipid surface promotes the forma tion of a high affinity complex of apoC-II and LpL, activation occurs via d irect helix-helix interactions between apoC-II39-62 and the loop covering t he active site of LpL.