Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A(2)
Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A(2)
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DOI：
10.1093/emboj/16.3.651
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发表时间：
1997-02-03
期刊：
影响因子：
11.4
通讯作者：
Wang, CC
中科院分区：
文献类型：
--
作者：
Yao, Y;Zhou, YC;Wang, CC
酸性磷脂酶A（2）（APLA（2））是一种含有7个二硫键的蛋白质，在盐酸胍中还原变性后，其自发复性率很低。在含有GSH和GSSG的氧化还原缓冲液中，蛋白质二硫键异构酶（PDI）能显著提高APLA（2）的复性率，并阻止APLA（2）在复性过程中的聚集。S-甲基化PDI（mPDI）不具有异构酶，但与天然PDI一样具有几乎完全的分子伴侣活性，对APLA的再活化或聚集没有影响（2）。然而，与APLA（2）的摩尔比分别为0.1和0.9的PDI和mPDI的同时存在完全再活化了变性的酶，如比率为1的单独的PDI那样。在比率分别为0.1和0.15时，它们完全抑制APLA（2）聚集，如比率为0.25时单独的PDI一样。此外，延迟向重折叠缓冲液中加入PDI大大降低了APLA（2）的再活化产率，但这种恶化效应可以通过重折叠缓冲液中存在mPDI而显著减轻。mPDI可阻止APLA（2）在复性过程中的聚集。有人提出，PDI作为折叠酶的体外作用由异构酶和伴侣活性组成，后者的活性可被mPDI完全取代。
The spontaneous reactivation yield of acidic phospholipase A(2) (APLA(2)), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low, Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA(2) during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA(2). However, the simultaneous presence of PDI and mPDI in molar ratios to APLA(2) of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA(2) aggregation, as does PDI alone at a ratio of 0.25, Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA(2), but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer, Without GSSG, mPDI prevents the aggregation of APLA(2) during refolding, It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.