Interaction of an Apolipoprotein (ApoLP-Alanine) with Phosphatidylcholine

The apolipoprotein containing C-terminal alanine (apoLP-Ala) from very low density lipoprotein and phosphatidylcholine were used as a prototype to study lipid-protein interactions in human plasma lipoproteins. ApoLP-Ala strongly inhibited the reactivation of delipidated mitochondrial β-hydroxybutyrate dehydrogenase, an enzyme which requires phosphatidylcholine for biological activity. When apoLP-Ala was sonicated with a 100-fold molar excess of phosphatidylcholine, a lipid-protein complex resulted which could be isolated free of excess lipid by ultracentrifugal flotation in potassium bromide solution at a density of 1.063-1.21 g/ml. The complex contained an average of 38 ± 5 phosphatidylcholine molecules for every 1 apoLP-Ala molecule. Further experiments showed that ultracentrifugation on a sucrose density gradient afforded a heterogeneous population of complexes whose average stoichiometry was 46 phosphatidylcholine molecules per apoLP-Ala molecule. When a saline gradient was used, the average stoichiometry was reduced to 30:1. When the titration of apoLP-Ala with a sonicated dispersion of phosphatidylcholine was observed by circular dichroism, the calculated α-helical content of the protein increased from 22 to 54%. At maximal helicity, the average stoichiometry of the complex was about 50 phosphatidylcholine molecules to 1 apoLP-Ala molecule. When the titration experiment was monitored by intrinsic fluorescence of the tryptophan residues, the maximum at 352 nm was gradually blue-shifted until a level of about 80 phosphatidylcholine molecules to 1 apoLP-Ala molecule was reached. These studies indicate that apoLP-Ala can bind up to a saturating level of 50-80 phosphatidylcholine molecules. The binding of phosphatidylcholine induces a shift from a disordered to a helical secondary structure and shifts one or more of the three tryptophan residues from a more polar to a more hydrophobic environment. These results show that highly lipidated species of apoLP-Ala may be formed which can be partially dissociated at high salt concentrations and suggest that ionic associations of lipid and apolipoproteins may play at least a minor role in the formation of plasma lipoprotein complexes. Our experiments are discussed in terms of their relationship to possible lipid-protein interactions in membranes.