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Isolation and characterization of apoLp-Gln-II (apoA-II), a plasma high density apolipoprotein containing two identical polypeptide chains.

S. Lux,K. John,H. Brewer

Published 1972 in Journal of Biological Chemistry

ABSTRACT

Abstract 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 m guanidine 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. These studies indicate that apoLp-Gln-II is composed of two identical polypeptide chains connected by a single disulfide bridge. Each chain has NH2-terminal pyrrolidonecarboxylic acid and COOH-terminal glutamine. The composition of the reduced and S-carboxymethylated chains is: Cys(Cm)1, PCA1, Asp2, Asn1, Thr6, Glu8, Gln7, Pro4, Gly3, Ala5, Val6, Met1, Ile1, Leu8, Tyr4, Phe4, and Lys9. Each polypeptide chain contains 77 residues and has a molecular weight of 8690.

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