Human Plasma High Density Lipoprotein: INTERACTION OF THE CYANOGEN BROMIDE FRAGMENTS FROM APOLIPOPROTEIN GLUTAMINE II (A-II) WITH PHOSPHATIDYLCHOLINE

Abstract

Apolipoprotein-glutamine-II (apoLP-Gln-II) is one of the two major protein constituents of human plasma high density lipoproteins (HDL). ApoLP-Gln-II contains two identical chains of 77 amino acids each, which are linked by a single disulfide bond at residue 6. Each chain contains a single methionine at position 26. In the present study we have examined the phospholipid binding properties of apoLP-Gln-II, of the NH2-terminal cyanogen bromide fragment both in the reduced form (CNBr IV, residues 1 to 26) and with the disulfide bond intact (CNBr IV)2, of the COOH-terminal cyanogen bromide fragment (CNBr III, residues 27 to 77) and of performic acid-oxidized apoLP-Gln-II. The binding of phosphatidylcholine was monitored by the inhibition of the reactivation of delipidated β-hydroxybutyrate dehydrogenase from beef heart mitochondria and by the formation of phospholipid-protein complexes which were isolated by density gradient ultracentrifugation in sucrose. The circular dichroism and immunochemical properties of these fragments and derivatives of apoLP-Gln-II were studied both in the presence and absence of phosphatidylcholine. The results may be summarized as follows. (a) ApoLP-Gln-II and its performic acid-oxidized derivative were potent inhibitors of the reactivation of β-hydroxybutyrate apodehydrogenase. One microgram of these preparations gave approximately 50% inhibition. The COOH-terminal CNBr fragment, CNBr III, also inhibited reactivation of the apodehydrogenase at low concentrations, but was less effective than the intact protein. Both CNBr IV and (CNBr IV)2 failed to inhibit reactivation of the dehydrogenase. (b) ApoLP-Gln-II, performic acid-oxidized apoLP-Gln-II and CNBr III formed complexes with phosphatidylcholine which were isolated by density gradient ultracentrifugation in sucrose (d 1.063 to 1.210). These isolated complexes contained, respectively, 1.16, 1.56, and 2.00 mg of phosphatidylcholine per mg of protein or peptide. When (CNBr IV)2 was reconstituted with phosphatidylcholine and subjected to ultracentrifugation under identical conditions, the isolated peptide contained only 0.08 mg of phospholipid per mg of peptide. (c) Reconstitution with phosphatidylcholine resulted in an apparent increase in α helical content (as judged by an increase in θ222 nm in the circular dichroism spectrum). The magnitude of the changes were as follows: 49% to 64% for apoLP-Gln-II, 32 to 57% for performic acid-oxidized apoLP-Gln-II, and 32 to 44% for CNBr III. There was no significant change in the CD spectra of (CNBr IV)2 detected in the presence of phosphatidylcholine. (d) Performic acid oxidation of apoLP-Gln-II did not alter the qualitative immunoprecipitin lines when this antigen was tested against rabbit antisera prepared against apoLP-Gln-II. The same antisera reacted with CNBr III and (CNBr IV)2, but formed lines of only partial identity between the fragments or between each fragment and apoLP-Gln-II. Rabbit antisera to CNBr III formed precipitin lines of complete identity between this fragment, apoLP-Gln-II, and performic acid-oxidized apoLP-Gln-II. No differences were noted after reconstitution with phosphatidylcholine. Anti-CNBr III did not form detectable precipitin lines with (CNBr IV)2. By the tests we have employed, we conclude that CNBr III or the COOH-terminal two-thirds of apoLP-Gln-II exhibits preferential interaction with phosphatidylcholine, although to a lesser extent than does the intact protein. The NH2-terminal fragment did not show a significant interaction with phosphatidylcholine whether the disulfide linkage was intact or not. Modification of the disulfide linkage or of the methionine at residue 26 did not alter the qualitative immunoprecipitin test with the antisera employed.