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J. Biol. Chem., Vol. 280, Issue 40, 33766-33774, October 7, 2005

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Glutamate Cysteine Ligase Catalysis

DEPENDENCE ON ATP AND MODIFIER SUBUNIT FOR REGULATION OF TISSUE GLUTATHIONE LEVELS^*

From the Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056

Glutamate cysteine ligase (GCL), which synthesizes -glutamyl-cysteine (-GC), is the rate-limiting enzyme in GSH biosynthesis. -GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(–/–) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(–/–) mice to examine the role of GCLM on -GC synthesis by GCLholo. GCLM decreased the K_m for ATP by 6-fold and, similar to other species, decreased the K_m for glutamate and increased the K_i for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the K_cat for -GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for -GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase -GC synthesis. On the other hand, our results from kidney suggest that -GC synthesis may be controlled post-translationally.

Received for publication, April 27, 2005 , and in revised form, August 3, 2005.

^* This work was supported by National Institutes of Health Grants R01 ES012463 (to T. P. D.) and R01 ES10133 (to H. G. S.) and the Center for Environmental Genetics P30 ES06096 (to T. P. D., H. G. S., and D. W. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 513-558-0373; Fax: 513-558-0925;E-mail: daltontp{at}ucmail.uc.edu.

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