Co-purification of mitochondrial HSP70 and mature proteins disulfide isomerase with a functional rat kidney high-M-r cysteine S-conjugate beta-lyase

S-(1,1,2,2-Tetrafluoroethyl)-L-Cysteine (TFEC, the cysteine S-conjugate of tetrafluoroethylene) is an example of a nephrotoxic, halogenated cysteine S -conjugate. Toxicity results in part from the cysteine S-conjugate beta -ly ase(s)-catalyzed conversion of TFEC to a thioacylating fragment with the as sociated production of pyruvate and ammonia. In the present study, we have demonstrated that rat kidney homogenates contain at least three enzyme frac tions that are capable of catalyzing a cysteine S.-conjugate beta -lyase re action with TFEC. One of these fractions contains a high-M-r lyase. At leas t two proteins co-purify with this high-M-r complex. N-Terminal analysis (1 5 cycles) revealed that the smaller species was mature protein disulfide is omerase (M-r similar to 54,200) from which the 24 amino acid endoplasmic re ticulum signal peptide had been removed. Internal amino acid sequencing (15 cycles) revealed that the larger species was mitochondrial HSP70 (mtHSP70; M-r similar to 75,000). The present findings offer an explanation for the previous observation that mtHSP70 in kidney mitochondria is heavily thioacy lated when rats are injected with TFEC (Bruschi et al., J Biol Chem 1993;26 8:23157-61). (C) 2001 Elsevier Science Inc. All rights reserved.