A NONRADIOACTIVE ASSAY FOR MICROSOMAL CYSTEINE-S-CONJUGATE N-ACETYLTRANSFERASE ACTIVITY BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

Microsomal cysteine-S-conjugate N-acetyltransferase, an enzyme specifi c for S-substituted cysteines, plays an important role in the detoxica tive metabolism of xenobiotics by catalyzing the N-acetylation of cyst eine-S-conjugates. Cysteine-S-conjugate N-acetyltransferase activity i s generally assayed by measuring the amount of N-[C-14]acetyl-S-benzyl -L-cysteine generated from the model compound S-benzyl-L-cysteine and [C-14]acetyl-CoA and subsequent extraction of the product. Although se nsitive, this method is costly and time consuming. For safety and envi ronmental reasons we developed a nonradioactive assay for cysteine-S-c onjugate N-acetyltransferase activity. Our method depends upon the ace tylation of the uv-sensitive model compound 4-nitro-S-benzyl-L-cystein e. The test mixture is separated by HPLC, guaranteeing that no byprodu cts interfere with the determination of product formation. Radioactive and nonradioactive methods were compared using different porcine kidn ey samples. With the nonradioactive test we determined Values of K-m a nd V-max of both 4-nitro-S-benzyl-L-cysteine and acetyl-CoA. In summar y, this new nonradioactive assay is sensitive, less costly, safer, les s time-consuming, and less laborious than radioactive assays for cyste ine-S-conjugate N-acetyltransferase. (C) 1994 Academic Press, Inc.