Abstract
The submitochondrial localization and identity of enzymes which metabolize cysteine conjugates were investigated. Glutamine transaminase K was purified from rat kidney mitochondrial soluble fraction and was shown to be a cysteine conjugate beta-lyase. The purified mitochondrial enzyme is similar to the cytosolic glutamine transaminase K whose beta-lyase activity with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is regulated by concurrent transamination (Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) J. Biol. Chem. 261, 15529-15537). However, beta-lyase activity in whole mitochondria is largely independent of regulation by cosubstrates for transamination suggesting that factors present in mitochondria are able to support the beta-lyase activity in the absence of added alpha-keto acid. Fractionation of mitochondria results in a loss of the independent beta-lyase activity. However, the majority of the beta-lyase activity can be recovered in the matrix if it is stimulated by the addition of alpha-keto-gamma-methiolbutyrate. The data suggest that the regulation of beta-elimination by the competing transamination pathway is different for each substrate and that multiple beta-lyases may exist in rat kidney. S-(2-Benzothiazolyl)-L-cysteine (BTC) is a poor substrate for purified glutamine transaminase K from mitochondria and cytosol, but BTC is as active as DCVC in crude mitochondrial matrix suggesting that other enzymes may be present. In contrast to DCVC, with BTC as substrate, the beta-lyase activity of the purified enzyme and enzyme(s) in the mitochondrial matrix is largely alpha-keto acid-independent. The existence of multiple enzymes is also supported by the observation that alpha-keto acids which are not substrates for purified glutamine transaminase K from mitochondria and cytosol do stimulate beta-lyase activity in the mitochondrial matrix fraction. Mitoplasts were found to be sensitive to DCVC toxicity consistent with the matrix localization of beta-lyase activity. The possible role in cysteine conjugate toxicity of matrix enzyme regulation by alpha-keto acids is discussed.
Highlights
P-lyase, a pyridoxal phosphate(PLP)’-dependent enzyme (Equation 1 (3-19)).This mechanism was first proposed by Schultzeand co-workers (6,7 ) who suggested that S-(1,2dichloroviny1)-L-cysteine (DCVC) is metabolized by P-elimination (Equation 1)to a reactive thiol which binds covalently to cellular macromolecules
The role in toxicity of @-lyasesfrom different mito- activity we investigated the submitochondrial distributionof chondrial compartments is investigatwedith particular atten- the activity andcompared it to the distributionof mitochontion to the participation of mitochondrial glutamine trans- drial glutamine transaminase K (Table 2)
Metabolism of DCVC and the Covalent Binding of Reactiue bothcysteine conjugate@-lyase activity and mitochondrial Species in Mitochondria and Mitoplasts-Initially, we inves- glutamine transaminaseK activity were located in the matrix tigated the metabolism of [“C]DCVC in mitochondria and fraction and thawt hen mitochondria are fractionated, factors mitoplasts in the presence and absence of added a-keto-y- are lost that support the @-lyase activity
Summary
Vol 263, No 7, Issue of March 5,pp. 3395-3401, 1988 Printed in U.S.A. The Role of Mitochondrial Matrix Enzymes in theMetabolism and Toxicity of Cysteine Conjugates*. Metabolism of DCVC and the Covalent Binding of Reactiue bothcysteine conjugate@-lyase activity and mitochondrial Species in Mitochondria and Mitoplasts-Initially, we inves- glutamine transaminaseK activity were located in the matrix tigated the metabolism of [“C]DCVC in mitochondria and fraction and thawt hen mitochondria are fractionated, factors mitoplasts in the presence and absence of added a-keto-y- are lost that support the @-lyase activity These factors can methiolbutyrate (MTB) (Table 1). @-Lyase activity ization of Its Role as a @-Lyase-Since cytosolic glutamine in mitochondria was stimulated only 60% by MTB while in transaminase K and mitochondrial glutamine transaminase mitoplasts the activity increase4d-fold (Table 1).The differ- K have similar substrate specificity and physical properties ence instimulation was dueto a loss of activityinthe (20, 21), it seemed that the matrix activity might be due to mitoplasts when no MTB was added to the incubations. The mitochondria are shown as 100% in both cases, the value in the absence of MTB is 2-fold less than in the presence of MTB (Table 1).Typically 250-400 mg of protein were used for one fractionation experiment, and the data are the meanf S.D. for four independent fractionations from pooled kidneys of four to eight rats
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