Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney.
Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N'-bis-(3-D-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immuno-affinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and K(m) values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.
Aigner
A
A
Institut für Biochemie, Technische Hochschule Darmstadt, Germany.
Jäger
M
M
Pasternack
R
R
Weber
P
P
Wienke
D
D
Wolf
S
S
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D011339:Probenecid / Q000494:pharmacology; D012076:Renal Agents; D013552:Swine; D015262:Xenobiotics / Q000378:metabolism
D000596:Amino Acids; D015853:Cysteine Proteinase Inhibitors; D012076:Renal Agents; D015262:Xenobiotics; D000123:Acetyltransferases; C034954:cysteine S-conjugate N-acetyltransferase; D011339:Probenecid