Hydroxylamine treatment differentially inactivates purified rat hepatic asialoglycoprotein receptors and distinguishes two receptor populations.
Hydroxylamine treatment differentially inactivates purified rat hepatic asialoglycoprotein receptors and distinguishes two receptor populations.
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羟胺处理差异性地灭活纯化的大鼠肝脱唾液酸糖蛋白受体并区分两个受体群体。
DOI：
10.1074/jbc.270.36.21388
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Weigel,PH
中科院分区：
文献类型：
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作者：
Zeng,FY;Weigel,PH
我们以前表明，两个亚群的去唾液酸糖蛋白受体（ASGP-Rs），指定状态1和状态2 ASGP-Rs，存在于完整的细胞和状态2 ASGP-Rs可以在渗透性大鼠肝细胞中失活的温度和ATP依赖性的方式。这些失活的ASGP-R可以通过添加棕榈酰-CoA定量地再活化（Weigel，P.H.，和Oka，J.A.（1993）J.Biol.Chem.268，27186-27190）。在这里，我们表明，约50%的纯化的大鼠ASGP-Rs被灭活的羟胺在温和的条件下处理。亲和纯化的ASGP-Rs的活性通过在固定到硝酸纤维素上后在斑点印迹分析中测量125 I-去唾液酸-乳清类粘蛋白（ASOR）的特异性结合来评估。在4°C下用0.0125-1.0 M NH 2 OH（pH 7.4）处理溶液中的ASGP-Rs 4 h导致ASOR结合活性的逐渐丧失。ASGP-R失活与NH 2 OH更容易发生在碱性pH值或在室温下。用Tris进行类似处理对ASGP-R活性没有影响。ASGP-R活性损失的动力学和这种失活的剂量反应都是双相的，表明存在两个相等的ASGP-R群体，对NH 2 OH具有不同的敏感性。通过用0.2 M NH 2 OH（4°C，4 h）或1.0 M NH 2 OH（4 ° C，1 h）处理使ASGP-Rs的更敏感群体（~50%）失活，如通过SDS-聚丙烯酰胺凝胶电泳评估的，没有可检测的肽裂解。从氯喹或莫能菌素处理的肝细胞中纯化的状态1 ASGP-Rs对NH 2 OH处理的敏感性显着降低（动力学和剂量依赖性）。此外，在温和条件下，NH 2 OH导致约50%的预结合至ASOR-琼脂糖凝胶的总ASGP-R（状态1和状态2）的解离和失活，而相同的处理导致仅<20%的状态1 ASGP-R从这种预形成的复合物中解离。如所附论文（Zeng，F.是的，Kaphalia，B.美国，安萨里湾A.美国，和Weigel，P. H.（1995）J.Biol.Chem.270，21382-21387）活性ASGP-Rs的所有三个RHL亚基实际上都含有共价连接的棕榈酸酯和硬脂酸酯。在培养的细胞中，[3 H]棕榈酸通过代谢结合到所有三个亚基中。这些放射性标记的脂肪酸通过温和的NH 2 OH处理从纯化的ASGP-Rs中完全释放。我们得出结论，ASGP-Rs的NH 2 OH敏感的亚群对应于先前描述的状态2 ASGP-Rs，这些受体需要脂肪酰化的配体结合活性。
We previously showed that two subpopulations of asialoglycoprotein receptors (ASGP-Rs), designated State 1 and State 2 ASGP-Rs, are present in intact cells and that State 2 ASGP-Rs can be inactivated in permeable rat hepatocytes in a temperature- and ATP-dependent manner. These inactivated ASGP-Rs can be quantitatively reactivated by the addition of palmitoyl-CoA (Weigel, P. H., and Oka, J. A.(1993)J. Biol. Chem.268, 27186-27190). Here we show that ~50% of purified rat ASGP-Rs are inactivated by treatment with hydroxylamine under mild conditions. The activity of affinitypurified ASGP-Rs was assessed by measuring the specific binding of125I-asialo-orosomucoid (ASOR) in a dot-blot assay after immobilization onto nitrocellulose. Treatment of ASGP-Rs in solution with 0.0125-1.0 M NH2OH, pH 7.4, at 4°C for 4 h resulted in a progressive loss of ASOR binding activity. ASGP-R inactivation with NH2OH occurred more readily at basic pH or at room temperature. Similar treatment with Tris had no effect on ASGP-R activity. The kinetics of ASGP-R activity loss and the dose-response for this inactivation were both biphasic, indicating the presence of two equal populations of ASGP-Rs with different sensitivities to NH2OH. The more sensitive population of ASGP-Rs (~50%) was inactivated by treatment with 0.2 M NH2OH (4°C, 4 h) or with 1.0 M NH2OH (4°C, 1 h) without detectable peptide cleavage as assessed by SDS-polyacrylamide gel electrophoresis. State 1 ASGP-Rs, purified from chloroquine- or monensin-treated hepatocytes, showed significantly less sensitivity to NH2OH treatment (both in kinetics and dose dependence). Furthermore, under mild conditions NH2OH caused dissociation and inactivation of ~50% of the total ASGP-Rs (State 1 and State 2) that were prebound to ASOR-Sepharose, whereas the same treatment caused dissociation of only <20% of State 1 ASGP-Rs from such preformed complexes. As shown in the accompanying paper (Zeng, F. Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H.(1995)J. Biol. Chem.270, 21382-21387) all three RHL subunits of active ASGP-Rs, in fact, contain covalently attached palmitate and stearate. In cultured cells, [3H]palmitic acid is metabolically incorporated into all three subunits. These radiolabeled fatty acids are completely released from purified ASGP-Rs by mild NH2OH treatment. We conclude that the NH2OH-sensitive subpopulation of ASGP-Rs corresponds to the previously described State 2 ASGP-Rs and that these receptors require fatty acylation for their ligand binding activity.