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.

Green urine in a critically ill patient.

The development of discolored urine in the critically ill patient, although uncommon, may have many possible causes, with the most likely source related to medication administration. Studies were undertaken in a 39-year-old man who developed dark green urine while in the intensive care unit for neutropenic sepsis. Although the patient had developed prior nonoliguric renal failure stemming from his sepsis, his renal function at the time of presentation of urine discoloration was considered normal. Review of his medications and intravenous infusions suggested the most likely cause was the food dye placed in his enteral tube feedings. Spectrophotometric evaluation of the urine confirmed the presence of Food Dye and Color Blue Number 1 (FD&C Blue No. 1). This case shows that significant gastrointestinal absorption of FD&C Blue No. 1 can occur. FD&C Blue No. 1 should be considered in the differential diagnosis of dark green discolored urine.

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter.

Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1 proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.