Structural characterization, tissue distribution, and functional expression of murine aminoacylase III.
Many xenobiotics are detoxified through the mercapturate metabolic pathway. The final product of the pathway, mercapturic acids (N-acetylcysteine S-conjugates), are secreted predominantly by renal proximal tubules. Mercapturic acids may undergo a transformation mediated by aminoacylases and cysteine S-conjugate beta-lyases that leads to nephrotoxic reactive thiol formation. The deacetylation of cysteine S-conjugates of N-acyl aromatic amino acids is thought to be mediated by an aminoacylase whose molecular identity has not been determined. In the present study, we cloned aminoacylase III, which likely mediates this process in vivo, and characterized its function and structure. The enzyme consists of 318 amino acids and has a molecular mass (determined by SDS-PAGE) of approximately 35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is approximately 140 kDa as determined by size-exclusion chromatography, which suggests that it is a tetramer. In agreement with this hypothesis, transmission electron microscopy and image analysis of aminoacylase III showed that the monomers of the enzyme are arranged with a fourfold rotational symmetry. Northern analysis demonstrated an approximately 1.4-kb transcript that was expressed predominantly in kidney and showed less expression in liver, heart, small intestine, brain, lung, testis, and stomach. In kidney, aminoacylase III was immunolocalized predominantly to the apical domain of S1 proximal tubules and the cytoplasm of S2 and S3 proximal tubules. The data suggest that in kidney proximal tubules, aminoacylase III plays an important role in deacetylating mercapturic acids. The predominant cytoplasmic localization of aminoacylase III may explain the greater sensitivity of the proximal straight tubule to the nephrotoxicity of mercapturic acids.
Pushkin
Alexander
A
Division of Neurology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1689, USA. apushkin@mednet.ucla.edu
Carpenito
Gerardo
G
Abuladze
Natalia
N
Newman
Debra
D
Tsuprun
Vladimir
V
Ryazantsev
Sergey
S
Motemoturu
Srilakshmi
S
Sassani
Pakan
P
Solovieva
Nadezhda
N
Dukkipati
Ramnath
R
Kurtz
Ira
I
D000111:Acetylcysteine / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary
D000581:Amidohydrolases; C483737:aminoacylase III, mouse; D000111:Acetylcysteine