McConathy W J, Alaupovic P
Biochemistry. 1976 Feb 10;15(3):515-20. doi: 10.1021/bi00648a010.
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.
本报告描述了对载脂蛋白D（ApoD）特性的进一步研究，ApoD是一种最近才被认识的人血浆载脂蛋白，并给出了其脂蛋白形式即脂蛋白D（LP-D）的分离和分布结果。通过结合羟基磷灰石和葡聚糖G - 100柱层析的方法分离得到的ApoD，在7%聚丙烯酰胺凝胶上迁移时呈现为单一条带，其迁移率介于A-II和C-II多肽之间。在双向扩散和免疫电泳中，ApoD仅与抗ApoD血清发生反应。它的特征是含有包括半胱氨酸在内的所有常见氨基酸。氨基末端氨基酸被封闭。碳水化合物分析表明，ApoD是一种糖蛋白，其中葡萄糖、甘露糖、半乳糖、氨基葡萄糖和唾液酸占ApoD干重的18%。ApoD的估计分子量为22100。ApoD在血清中以脂蛋白形式存在，可通过两种不同的层析方法从高密度脂蛋白3中分离得到。在第一种方法中，高密度脂蛋白3用神经氨酸酶处理后在伴刀豆球蛋白A上进行层析。含有LP-D的保留部分通过羟基磷灰石柱层析进行纯化。或者，通过将高密度脂蛋白3或全血清在含有抗ApoD抗体的免疫吸附剂上进行层析，再结合羟基磷灰石柱层析的方法来分离LP-D。LP-D在分析超速离心中显示出单一、对称的边界，在7%聚丙烯酰胺凝胶电泳上呈现为单一一条带。将其注射到兔子体内可产生仅与ApoD发生反应的抗血清。在免疫电泳中，LP-D的迁移率与脂蛋白A（LP-A）不同。LP-D和LP-A的直接免疫学比较显示二者无同一性反应。LP-D由65 - 75%的蛋白质和25 - 35%的脂质组成。脂质部分含有胆固醇、胆固醇酯、甘油三酯和磷脂。磷脂组成的特征是溶血卵磷脂和鞘磷脂含量相对较高，而卵磷脂含量相对较低。我们从这些研究中得出结论，ApoD是一种独特的载脂蛋白，以一种独特的脂蛋白家族形式存在，其大分子分布从极低密度脂蛋白延伸到极高密度脂蛋白，但在高密度脂蛋白3中浓度最高，在高密度脂蛋白中浓度最低。
