Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants.
The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.
D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D005260:Female; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008081:Liposomes; D008892:Milk / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D008081:Liposomes; D010713:Phosphatidylcholines; C047037:1,2-ditetradecylphosphatidylcholine; D008071:Lipoprotein Lipase