Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.
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.
Yao
Y
Y
The National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Zhou
Y
Y
Wang
C
C
D017836:Agkistrodon / Q000378:metabolism*; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000378:metabolism*; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D012542:Scattering, Radiation
D003435:Crotalid Venoms; D004220:Disulfides; D006146:Guanidines; D018832:Molecular Chaperones; D010741:Phospholipases A; D054467:Phospholipases A2; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019791:Guanidine; D004229:Dithiothreitol