PURIFICATION AND CHARACTERIZATION OF CYSTEINE-S-CONJUGATE N-ACETYLTRANSFERASE FROM PIG-KIDNEY

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-ac etylation of various S-substituted cysteines in liver and kidney. We d escribe here the purification and more detailed characterization of th is enzyme catalysing the final reaction of mercapturic acid biosynthes is, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetylt ransferase by deoxy-BIGCHAP [N,N'-bis-(3-D-gluconamidopropyl)deoxychol amide] was the prerequisite for partial purification by means of anion -exchange chromatography. The molecular mass of the enzyme was determi ned by gel filtration. A polyclonal antiserum was raised against the e xcised protein band from SDS/PAGE and purified antibodies were used fo r the complete purification of native cysteine-S-conjugate N-acetyltra nsferase by immunoaffinity chromatography. A dimeric form of the enzym e was sometimes detected on SDS/PAGE, depending on the degree of purif ication. For further characterization of cysteine-S-conjugate N-acetyl transferase, the stability of catalytic activity, the pH optimum and K -m values were determined. The inhibitory effects of various agents we re tested, revealing a substantial, yet not complete, loss of cysteine -S-conjugate N-acetyltransferase activity after treatment with cystein e proteinase inhibitors or probenecid under various conditions.