The Interaction of Apolipoprotein-Serine with Phosphatidylcholine

Apolipoprotein-serine (apoLP-Ser) is one of the protein constituents of human plasma very low density lipoproteins (VLDL). The protein has 57 amino acids with 1 residue each of methionine and tryptophan. Cleavage of apoLP-Ser with cyanogen bromide yields the NH2-terminal fragment (CNBr I) and COOH-terminal fragment (CNBr II) having 38 and 19 residues, respectively. CNBr II contains the single residue of tryptophan. We have studied the interaction of egg phosphatidylcholine with apoLP-Ser and its cyanogen bromide fragments. The binding of phospholipid was studied in the following ways: (a) by inhibition of the reactivation of defatted β-hydroxybutyrate dehydrogenase from beef heart mitochondria; (b) by changes in circular dichroic and fluorescence spectra; (c) by isolation of complexes of the protein and peptide fragments with phospholipid, using density gradients of KBr; and (d) by rouleaux formation by phospholipid vesicles as determined by electron microscopy after negative staining with phosphotungstic acid. ApoLP-Ser strongly inhibited the reactivation of defatted β-hydroxybutyrate dehydrogenase; 10 µg of the apoprotein inhibited reactivation by 85%. CNBr I and CNBr II inhibited reactivation to a lesser extent than did the intact apoprotein. The α helical content of apoLP-Ser was about 56%, as determined by circular dichroism. Mixing the apoprotein with bilamellar vesicles of phosphatidylcholine resulted in an estimated increase in the α helical content to about 73%. The apparent helicity of CNBr I was increased from 34 to 65% and of CNBr II from 15 to 25% in the presence of phosphatidylcholine. The addition of phospholipid vesicles to apoLP-Ser produced a blue shift (6.0 nm) in the fluorescence maximum, indicating a shift of the tryptophan to a more hydrophobic environment. A similar finding was made with CNBr II in the presence of phospholipid. In ultracentrifugal flotation experiments, complexes of the phospholipid with apoLP-Ser, CNBr I, and CNBr II, each floated at a density less than 1.08 g per ml, whereas apoLP-Ser in the absence of phospholipid sedimented to a density greater than 1.18 g per ml. Finally, when the phosphatidylcholine vesicles were titrated with increasing amounts of the protein or cyanogen bromide fragments and negatively stained with phosphotungstic acid, the vesicles exhibited a series of morphological changes from free vesicles to linear arrays of stacked discs as determined by electron microscopy. These findings are interpreted in light of a theory of phospholipid-binding involving amphipathic helical regions that contain positively and negatively charged amino acids and are consistent with the localization of such regions in the primary chemical structure of the molecule.