Abstract
Cysteine conjugate beta-lyase activity from rat kidney cortex was found in the cystosolic and mitochondrial fractions. With 2 mM S-(2-benzothiazolyl)-L-cysteine as the substrate, approximately two-thirds of the total beta-lyase activity was present in the cytosolic fraction. The kinetics of beta-lyase activity with three cysteine S-conjugates were different in the cytosolic and mitochondrial fractions, and the mitochondrial beta-lyase was much more sensitive to inhibition by aminooxyacetic acid than was the cytosolic activity. These results indicate that the beta-lyase activities in the two subcellular fractions are catalyzed by distinct enzymes. Nephrotoxic cysteine S-conjugates of halogenated hydrocarbons that require bioactivation by cysteine conjugate beta-lyase (S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, CTFC) were potent inhibitors of state 3 respiration in rat kidney mitochondria. Fractionation of mitochondria by digitonin treatment and comparison with marker enzyme distributions showed that the mitochondrial beta-lyase activity is localized in the outer mitochondrial membrane. Inhibition of the beta-lyase prevented the mitochondrial toxicity of DCVC and CTFC, and nonmetabolizable, alpha-methyl analogues of DCVC and CTFC were not toxic. Neither DCVC nor CTFC was toxic to mitoplasts, indicating that activation by the beta-lyase occurs on the outer membrane and may be essential for the expression of toxicity; in contrast, the direct acting nephrotoxin S-(2-chloroethyl)-DL-cysteine was toxic to both mitochondria and mitoplasts. Thus, the suborganelle localization of DCVC and CTFC bioactivation correlates with the observed pattern of toxicity.
Highlights
Cortex was found in the cystosolic and mitochondrial (EC 4.4.1.13), which catalyzes an a,@-eliminationto produce fractions
Previous studies with rat kidney slices and with rat kidney and liver mitochondria showed that mitochondrial respiration and 2-oxoacid dehydrogenases are Glutathione S-conjugates of several halogenated hydrocar- inhibited by DCVC [13,23,24,25], indicating that mitochondria bons are potent nephrotoxins [1].Renal metabolism of the may bethe target siteswithin the cell where DCVC exerts its glutathione conjugates yields the corresponding cysteine S- toxicity
Conjugates, which may be direct acting nephrotoxins or may The objective of the present work was to study the role of the renal mitochondrial @-lyasein cysteine S-conjugate-in
Summary
Materials-Digitonin (recrystallized twice from hot ethanol), aminooxyacetic acid, benzylamine HC1, Lubrol PX, rotenone, phenylmethylsulfonyl fluoride, a-keto-y-methiolbutyrate, glycylglycine,oxalacetic acid, ferricytochrome c (type VI), hexokinase (EC 2.7.1.1), respiration was measured according to the procedure of Estabrook [40] by the addition of 3.3 mM succinate and 0.3 mM ADP to either 1.5-3.0 mg of mitochondrial protein or 0.5-1.0 mg of mitoplast protein
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