Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma

Abstract

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.