Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure.
Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure.
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人α2-巨球蛋白半分子与纤溶酶的反应作为蛋白酶结合位点结构的探针。
DOI：
10.1021/bi00290a009
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Pizzo,SV
中科院分区：
文献类型：
--
作者：
Gonias,SL;Pizzo,SV
Steven L.摘要：人α 2-巨球蛋白（α 2 M）半分子是通过对天然蛋白进行有限还原和烷基化来制备的。与纤溶酶反应导致近定量切割的半分子Mr~180000亚基成Mr~ 90000片段。当纤溶酶与2 M全分子反应时，亚基裂解明显不完全。用放射性碘标记的蛋白酶比较了两种形式的α 2 M与纤溶酶和胰蛋白酶的结合能力。a2 M半分子结合等摩尔数的纤溶酶或胰蛋白酶。天然未还原的α 2 M结合的纤溶酶仅为胰蛋白酶的一半。这些数据与两个蛋白酶结合位点在天然α 2 M中相邻的假设一致。α 2 M半分子-纤溶酶复合物比半分子-胰蛋白酶复合物更不容易再结合，支持了这一解释。纤溶酶和α 2 M之间共价键形成的频率明显高于以前观察到的其他蛋白酶。与a2 M全分子或半分子反应的纤溶酶的大约80-90%变成共价结合。比较纯化的α 2 M-纤溶酶复合物与小底物和大底物的反应性。在22 ℃下测定了全分子-纤溶酶复合物和半分子-纤溶酶复合物水解HD-Val-Leu-Lys-p-硝基苯胺二盐酸盐的等效fccat/μ m值（分别为40 mM-1 s-1和39 mM-1 s-1，与游离纤溶酶测定的66 mM-1 s-1相比）。纤溶酶复合物和两种不同形式的a2 M以相当的速率消化纤维蛋白原（与游离纤溶酶相比缓慢）;然而，半分子纤溶酶复合物表现出与大豆胰蛋白酶抑制剂的反应性增加。α 2 M半分子-纤溶酶复合物从小鼠循环中迅速清除，反映受体结合和内吞作用。血浆蛋白酶抑制剂人α 2-巨球蛋白（α 2 M）是由四个等价的Mr~ 180000多肽链组成，这些多肽链通过二硫键结合成对。来自杜克大学医学中心病理学和生物化学系，达勒姆，北卡罗来纳州27710。收到于1983年4月6日。这项工作得到了国家心脏，肺和血液研究所资助HL 24066的支持。SLG是国家普通医学科学研究所（GM-07171）医学科学家培训计划奖的获得者。和整个分子通过强非共价相互作用（Swenson和霍华德，1979 a; Harpel，1973; Hall和Roberts，1978）。每摩尔的四聚体2 M可以结合至多2摩尔的α-胰凝乳蛋白酶或胰蛋白酶（Barrett等人，1979; Swenson &霍华德，1979 a; Pochon等人，1978），但只有1摩尔纤溶酶（Ganrot，1967 a; Gonias等，1982 a; Pochon等人，1978）或1摩尔合成的α-胰凝乳蛋白酶二聚体（Pochon等，1981年）。这种摩尔结合比的变化可以用一种a2 M结构模型来解释，
Steven L. Goniasand Salvatore V. Pizzo* abstract: Human a2-macroglobulin (a2M) half-molecules were prepared by limited reductionand alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr~180000 subunits into Mr~ 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with a2Mwhole mole-cules. The plasmin and trypsin binding capacities of the two forms of a2M were compared by using radioiodinated pro-teases. a2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced a2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native a2M. a2M half-molecule-plasmin com-plexes reassociated less readily than half-molecule-trypsin complexes, supporting thisinterpretation. The frequency of covalent bond formation between plasmin and a2M was con-siderably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with a2M whole molecules or half-molecules became covalently bound. The reactivities of purified a2M-plasmin complexes were compared with small and large substrates. Equivalent fccat/^ m values were determined at 22 C for the hydrolysis of HD-Val-Leu-Lys-p-nitroanilidedihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s’1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s'1 determined for free plasmin). Complexes of plasmin and the two different forms of a2M digested fibrinogen at comparable rates (slowly compared with free plasmin); however, thehalf-molecule-plasmin complex demonstrated increased reactivity with soybean trypsin inhibitor. a2M half-molecule-plasmin complex cleared rapidly from the cir-culation of mice, reflecting receptor binding and endocytosis. e plasma protease inhibitor human a2-macroglobulin (a2M) is composed of fourequivalent Mr~ 180 000 poly-peptide chains that are associated into pairs by disulfide bonds t From the Departments of Pathology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710. Received April 6, 1983. This work was supported by National Heart, Lung and Blood Institute Grant HL24066. SLG is a recipient of a Medical Scientist Training Program Award, National Institute of General Medical Sci-ences (GM-07171). and whole molecules by strong noncovalent interactions (Swenson & Howard, 1979a; Harpel, 1973; Hall & Roberts, 1978). Each mole of tetramerica2M can bind up to 2 mol of a-chymotrypsin or trypsin (Barrett et al., 1979; Swenson & Howard, 1979a; Pochon et al., 1978), but only 1 mol of plasmin (Ganrot, 1967a; Gonias et al., 1982a; Pochon et al., 1978) or 1 mol of a synthetic a-chymotrypsin dimer (Pochon et al., 1981). This variation in molar binding ratios may be explained by a model of a2M structure that includestwo