Herzig M C, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Biochemistry. 1990 Jul 10;29(27):6437-47. doi: 10.1021/bi00479a015.
We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.
我们通过用同双功能N-羟基琥珀酰亚胺（NHS）酯交联剂辛二酸二琥珀酰亚胺酯衍生化去唾液酸血清类黏蛋白（ASOR），开发了一种针对肝半乳糖基受体（GalR）的特异性化学亲和试剂[赫齐格，M.C.S.，& 韦格尔，P.H.（1989年）《生物化学》28卷，600页]。NHS-ASOR以30%-50%的效率与三个GalR亚基交联，这三个亚基被命名为大鼠肝凝集素（RHL）1、2和3。在此，我们研究了两个功能不同的GalR亚群（称为状态1或状态2 GalR）的表面受体和内部受体的亚基结构。新鲜分离的细胞，称为状态1细胞，在4℃下保存时，其表面仅表达活性状态1 GalR。当这些细胞在37℃下平衡时，它们的表面则同时表达状态1 GalR和状态2 GalR。这些细胞被称为状态1,2细胞。在用NHS-125I-ASOR在4℃下孵育后，状态1细胞或状态1,2细胞的表面或内部GalR将125I-ASOR掺入到所有三个RHL亚基中。通过SDS-PAGE放射自显影分析，所有条件下的放射性标记均相同，RHL 1:2:3的比例为1:1:1。还通过首先在4℃下将非放射性标记的NHS-ASOR与状态1或状态1,2肝细胞的表面或内部受体交联来研究天然GalR结构。然后用乙二醇双四乙酸（EGTA）洗涤这些细胞，用 Triton X-100提取，用抗血清类黏蛋白抗体进行免疫沉淀，并进行蛋白质印迹分析。对RHL 1或RHL 2/3特异的抗血清分别检测到分子量约为85K或约为90K - 115K的交联复合物以及未交联的天然亚基。在所有四种情况下，RHL 1的游离亚基与交联亚基的比例大于或等于5:1，RHL 2/3的比例小于或等于0.5:1。内化的GalR的游离亚基与交联亚基的比例与未内化的GalR相同。在将GalR与NHS/ASOR交联之前或之后消耗ATP也不会改变游离交联的RHL亚基的比例。我们得出结论，两个功能不同的GalR群体的表面和内部GalR具有相同的异源寡聚体亚基组成，并且这种GalR结构在胞吞作用或ATP消耗后仍然存在。
