Search

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)2failed 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 (d1.063 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)2was 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 nmin 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)2detected 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.

ApoLp-Gln-II (or apoA-II), one of the two major apolipoprotein components of human plasma high density lipoproteins, was subjected to enzymatic and nonenzymatic digestion with trypsin and cyanogen bromide. The individual peptides were isolated to homogeneity by chromatography on DEAE-cellulose, Cm-Sephadex, Sephadex G-100, Sephadex G-75, preparative thin layer plates, and by peptide mapping. Isolated peptides were assayed for purity by amino acid analysis, thin layer chromatography, polyacrylamide gel electrophoresis, and Edman NH2-terminal analysis. Cyanogen bromide cleavage followed by reduction and S-carboxymethylation produced two peptides, C-III and C-IV, of 51 and 26 residues, respectively. The smaller peptide (C-IV) contained homoserine and homoserine lactone and had NH2-terminal pyrrolidone carboxylic acid. It was the NH2-terminal cyanogen bromide peptide. This peptide contained the single S-carboxymethylcysteine residue. C-III was the COOH-terminal peptide. It contained COOH-terminal glutamine and a penultimate threonine residue. Tryptic digestion of reduced and alkylated apoLp-Gln-II yielded eight major tryptic peptides (T-I-1, T-I-2, T-I-3, T-III, T-IV, T-VI-1, T-VII, and T-VIII) and seven additional tryptic peptides which either contained or were contained in two or more of the eight major peptides. Only single unique NH2-terminal (T-I-2), COOH-terminal (T-VII), S-carboxymethylcysteine-containing (T-VIII), methionine-containing (T-VI-1), and isoleucine-containing (T-I-3) peptides were found. The combined compositions of the tryptic or cyanogen bromide peptides were equal to the composition of S-carboxymethyl-apoLp-Gln-II. These results confirm previous observations from this laboratory on intact and reduced and alkylated apoLp-Gln-II and support the concept that the molecule is composed of two identical chains of 77 residues each linked by a disulfide bond. The data available allow a partial alignment of these peptides and indicate that the disulfide bridge is in the NH2-terminal one-third of the molecule. The first three tryptic peptides in the sequence are T-I-2, T-VIII, and T-VI-1. T-VII is the COOH-terminal tryptic peptide. These assignments have been confirmed by manual and automated Edman degradations of the isolated tryptic and cyanogen bromide peptides performed in parallel with the present studies and published elsewhere (Brewer, H. B., Jr., Lux, S. E., Ronan, R., andJohn, K. M. (1972) Proc. Nat. Acad. Sci. U.S.A.69, 1304–1308).

ApoLp-Gln-II (or apoA-II), one of the two major apolipoproteins of human plasma high density lipoprotein, was isolated from a single female volunteer by DEAE-cellulose chromatography. The purified apoprotein was shown to be homogeneous by immunoelectrophoresis, polyacrylamide gel electrophoresis, and amino acid analysis. Distinctive features of the amino acid composition were the absence of histidine, arginine, and tryptophan. Reduction and carboxymethylation yielded 2 moles of S-carboxymethylcysteine per mole of intact apoLp-Gln-II. Few carboxymethyl groups could be added to apoLp-Gln-II prior to its reduction, indicating that cystine rather than cysteine was present in the intact apoprotein. This was confirmed by identification of cystine on enzymatic digestion of the intact apoprotein. Following reduction and S-carboxymethylation of apoLp-Gln-II, its molecular weight, as determined by gel filtration in 6 mguanidine hydrochloride, decreased from 16,700 ± 300 S.E.M. to 8,950 ± 150 S.E.M., indicating that apoLp-Gln-II is composed of two polypeptide chains of similar molecular weight. The two chains appeared to be identical by polyacrylamide gel electrophoresis in urea (pH 2.9 and 9.4), immunoelectrophoresis, and DEAE-cellulose chromatography. No NH2-terminal amino acid could be demonstrated by the dansyl chloride or phenylisothiocyanate methods, but digestion of apoLp-Gln-II with pyrrolidonecarboxylyl peptidase released pyrrolidonecarboxylic acid. Carboxypeptidase A digestion released glutamine and threonine. The kinetics of release as well as the inability to demonstrate threonine on hydrazinolysis indicated that glutamine was the COOH-terminal amino acid and threonine was the penultimate residue.