Co-purification of mitochondrial HSP70 and mature protein 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-lyase(s)-catalyzed conversion of TFEC to a thioacylating fragment with the associated production of pyruvate and ammonia. In the present study, we have demonstrated that rat kidney homogenates contain at least three enzyme fractions that are capable of catalyzing a cysteine S-conjugate beta-lyase reaction with TFEC. One of these fractions contains a high-M(r) lyase. At least two proteins co-purify with this high-M(r) complex. N-Terminal analysis (15 cycles) revealed that the smaller species was mature protein disulfide isomerase (M(r) approximately 54,200) from which the 24 amino acid endoplasmic reticulum signal peptide had been removed. Internal amino acid sequencing (15 cycles) revealed that the larger species was mitochondrial HSP70 (mtHSP70; M(r) approximately 75,000). The present findings offer an explanation for the previous observation that mtHSP70 in kidney mitochondria is heavily thioacylated when rats are injected with TFEC (Bruschi et al., J Biol Chem 1993;268:23157-61).
Cooper
A J
AJ
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY, USA. acooper@burke.org
Wang
J
J
Gartner
C A
CA
Bruschi
S A
SA
D000818:Animals; D013437:Carbon-Sulfur Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D020539:Sequence Analysis, Protein; D005466:Fluorocarbons
D013437:Carbon-Sulfur Lyases; D003545:Cysteine; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D006845:Hydrocarbons, Fluorinated; D019704:Protein Disulfide-Isomerases; C064116:S-(1,1,2,2-tetrafluoroethyl)cysteine; D005466:Fluorocarbons; C108902:cysteine-S-conjugate beta-lyase