Isolation, purification and identification of intracellular protein targets of ochratoxin A: mitochondrial aldehyde dehydrogenase class 2 is a high-affinity ochratoxin A-binding protein

Abstract

An affinity chromatography technique was utilised to isolate and purify the soluble intracellular protein targets of ochratoxin A (OA). OA was covalently immobilised on a beaded agarose matrix. Several proteins (approximately nine) in the soluble fraction of crude protein extracts of different bovine tissues were specifically bound to the immobilised OA. The binding of these proteins to the modified agarose could not be dissociated with mild buffer (buffer plus 0.5 M NaCl and 1% Tween 20), but the proteins were competitively eluted with free 25 mM OA in mild buffer. Only one major protein (55 kDa) remained tightly bound to the immobilised OA when the column was incubated with bovine liver extract and then washed extensively with higher concentrations of salt and detergent (2 M buffer plus NaCl and 5% Tween 20). The bound protein was eluted competitively with 25 mM OA and further purified using SDS-PAGE. The N-terminal sequence of 24 residues of the high-affinity OA-binding protein was shown to be AATQAVPTPNQQPEVLYNQIFINN. This sequence is identical to the 24 residues of the N-terminal sequence of bovine mitochondrial aldehyde dehydrogenase class 2 (ALDH2, EC 1.2.1.3). The molecular weight and tissue distribution of ALDH2 are the same as those of the high-affinity protein isolated in this study. Immobilised OA can serve as an excellent affinity matrix for the identification of target proteins of OA and for the purification of ALDH2 and possibly other proteins. © 1999 Society of Chemical Industry