Yao Y, Zhou Y, Wang C
The National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
EMBO J. 1997 Feb 3;16(3):651-8. doi: 10.1093/emboj/16.3.651.
The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), 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 APLA2 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 APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 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 APLA2 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 APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 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.
酸性磷脂酶A2（APLA2）是一种含有7个二硫键的蛋白质，在盐酸胍中还原和变性后，其自发重新激活产率非常低。蛋白质二硫键异构酶（PDI）显著提高重新激活产率，并在含有谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的氧化还原缓冲液中重折叠过程中防止APLA2聚集。S-甲基化的PDI（mPDI）没有异构酶活性，但具有与天然PDI几乎相同的伴侣活性，对APLA2的重新激活或聚集均无影响。然而，当PDI和mPDI分别与APLA2的摩尔比为0.1和0.9时，可使变性酶完全重新激活，这与单独使用摩尔比为1的PDI的效果相同。当它们与APLA2的摩尔比分别为0.1和0.15时，可完全抑制APLA2聚集，这与单独使用摩尔比为0.25的PDI的效果相同。此外，向重折叠缓冲液中延迟添加PDI会大大降低APLA2的重新激活产率，但重折叠缓冲液中存在mPDI可显著减轻这种恶化作用。在没有GSSG的情况下，mPDI可防止重折叠过程中APLA2的聚集。有人提出，PDI在体外作为折叠酶的作用包括异构酶和伴侣活性，而后者的活性可被mPDI完全替代。
