Rat Kidney Cytosol Research Articles

Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases.

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.

The effect of aristolochic acid I on expression of NAD(P)H:quinone oxidoreductase in mice and rats—A comparative study

Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H:quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined.We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver.NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Cefadroxil potency as cancer co-therapy candidate by glutathione s-transferase mechanism

Background: Glutathione S-transferases (GSTs) have an important role in the detoxification of electrophiles, such as some anticancer drugs. Compounds with phenolic and/or α,b-unsaturated carbonyl group have been known as GSTs inhibitor in vitro. Cefadroxil in vitro decreased GST-Pi activity but not GSTs in rat kidney cytosol. GST inhibitor in a specific organ and of a specific class is needed for safety in cancer chemotherapy. The study aims to observe the effect of cefadroxil on GSTs in vivo in rat kidney cytosol and then compare it to those seen for liver, lung, and spleen in vivo.

N-Biotinyl-S-(1,2-dichlorovinyl)-l-cysteine Sulfoxide as a Potential Model for S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide: Characterization of Stability and Reactivity with Glutathione and Kidney Proteins in Vitro

S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide (DCVCS) is a reactive and potent nephrotoxic metabolite of the human trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Because DCVCS covalent binding to kidney proteins likely plays a role in its nephrotoxicity, in this study biotin-tagged DCVCS, N-biotinyl-DCVCS (NB-DCVCS), was synthesized, and its stability in buffer alone and in the presence of rat blood or plasma was characterized in vitro. In addition, reactivity toward GSH and covalent binding to selected model enzymes and isolated kidney proteins were characterized. The half-lives of NB-DCVCS (39.6 min) and the DCVCS (diastereomer 1, 14.4 min; diastereomer 2, 6 min) in the presence of GSH were comparable. Incubating the model enzymes glutathione reductase and malate dehydrogenase with 10 μM NB-DCVCS for 3 h at 37 °C followed by immunoblotting using antibiotin antibodies demonstrated that glutathione reductase and malate dehydrogenase were extensively modified by NB-DCVCS. When rat kidney cytosol (6 μg/μL) was incubated with NB-DCVCS (312.5 nM to 5 μM) for 3 h at 37 °C followed by immunoblotting, a concentration-dependent increase in signal with multiple proteins with different molecular weights was observed, suggesting that NB-DCVCS binds to multiple kidney proteins with different selectivity. Incubating rat kidney cytosol with DCVCS (10-100 μM) prior to the addition of NB-DCVCS (2.5 μM) reduced the immunoblotting signal, suggesting that NB-DCVCS and DCVCS compete for the same binding sites. A comparison of the stability of NB-DCVCS and DCVCS in rat blood and plasma was determined in vitro, and NB-DCVCS exhibited higher stability than DCVCS in both media. Collectively, these results suggest that NB-DCVCS shows sufficient stability, reactivity, and selectivity to warrant further investigations into its possible use as a tool for future characterization of the role of covalent modification of renal proteins by DCVCS in nephrotoxicity.

14C-labeled Pulegone and Metabolites Binding to α2u-globulin in Kidneys of Male F-344 Rats*

Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and menthofuran were previously studied in rodents, and higher concentrations of pulegone- and menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, α2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 μCi/kg) or menthofuran (60 mg/kg, 120 μCi/kg) or menthones (80 mg/kg, 120 μCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney cytosol proteins of male but not female rats. The chemicals present in the male rat kidney cytosol after dialysis were extracted with dichloromethane and characterized by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). All parent compounds were detected, and the metabolites characterized included piperitone from pulegone or menthones treatment, menthones and possibly 8-hydroxymenthones from pulegone treatment, and mintlactones (diastereomeric mintlactone and isomintlactone) and 7a-hydroxymintlactone from menthofuran treatment. Analysis of the male rat kidney cytosol by a gel filtration column demonstrated that the retention was due to reversible binding of these chemicals with the male rat-specific protein α2u-globulin. However, binding of pulegone and/or metabolites to α2u-globulin did not produce accumulation of this protein in the kidney.

Transamination in the Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Due to the involvement of NDPS metabolism in its mechanism of toxicity, the detailed biotransformation of 14C-NDPS in rats was previously evaluated using high-performance liquid chromatography-electrospray ionization-mass spectrometry. In the present report, we describe the identification of two novel amino metabolites of NDPS, which were present in significant amounts in rat kidney tissues. Using liquid chromatography-tandem mass spectrometry and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (N-acetyl-2-NDASA). The mechanism of formation of 2-NDASA was studied in vitro. Incubations were carried out in rat liver and kidney cytosols using the major oxidative metabolite of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, as the substrate. Formation of 2-NDASA in vitro was confirmed using mass spectrometry. Inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (disulfiram) reduced 2-NDASA formation by 40 to 50%. Menadione (an inhibitor of aldehyde oxidase) and quercetin (an inhibitor of carbonyl reductase) did not show any effects. (Aminooxy)acetic acid, an inhibitor of pyridoxal 5'-phosphate-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using liquid chromatography-mass spectrometry, the transamination mechanism was further supported by the incorporation of a 15N-amino group in 2-NDASA when 15N-glutamic acid was included in the incubation mixture. Results from these studies show that transamination is a metabolic pathway in the clearance of NDPS in rats, and that cytosolic dehydrogenases and aminotransferases may be involved in this process.

Subcellular Distribution and Protein Binding of Perfluorooctanoic Acid in Rat Liver and Kidney

Perfluorooctanoic acid (PFOA) is an organic fluorochemical, and its elimination in rats is markedly sex-dependent. Liver and kidney are two primary tissues of distribution of PFOA in rats. In this study, the subcellular distribution of PFOA in male and female rat liver and kidney was examined. The results demonstrated that PFOA content in the liver cytosol of the female rat was significantly higher (49 ± 6% of total radioactive residues, TRR) than in the male liver (26 ± 5% TRR), whereas PFOA distribution in the heavier subcellular fractions, especially the nuclei and cell debris fraction, was marginally higher in male rat liver. In rat kidney, more than 70% of PFOA was distributed in the cytosolic fraction, with no significant difference between sexes. The degree of protein binding of PFOA in rat liver and kidney cytosol was analyzed by two different chromatographic methods. The percentage of protein-bound PFOA in the liver cytosol was found to be approximately 55% in both male and female rats. In contrast, significantly more PFOA was bound to cytosolic proteins in the kidney of male rats (42 ± 6% TRR) than in females (17 ± 5% TRR). Ligand blotting analysis revealed that multiple proteins from the liver cytosol, nuclei, and mitochondria fractions were capable of specific binding to PFOA.

Subcellular Distribution and Protein Binding of Perfluorooctanoic Acid in Rat Liver and Kidney

Perfluorooctanoic acid (PFOA) is an organic fluorochemical, and its elimination in rats is markedly sex-dependent. Liver and kidney are two primary tissues of distribution of PFOA in rats. In this study, the subcellular distribution of PFOA in male and female rat liver and kidney was examined. The results demonstrated that PFOA content in the liver cytosol of the female rat was significantly higher (49 ± 6% of total radioactive residues, TRR) than in the male liver (26 ± 5% TRR), whereas PFOA distribution in the heavier subcellular fractions, especially the nuclei and cell debris fraction, was marginally higher in male rat liver. In rat kidney, more than 70% of PFOA was distributed in the cytosolic fraction, with no significant difference between sexes. The degree of protein binding of PFOA in rat liver and kidney cytosol was analyzed by two different chromatographic methods. The percentage of protein-bound PFOA in the liver cytosol was found to be approximately 55% in both male and female rats. In contrast, significantly more PFOA was bound to cytosolic proteins in the kidney of male rats (42 ± 6% TRR) than in females (17 ± 5% TRR). Ligand blotting analysis revealed that multiple proteins from the liver cytosol, nuclei, and mitochondria fractions were capable of specific binding to PFOA.

Strain‐ and sex‐related differences of carbonyl reductase activities in kidney microsomes and cytosol of rats

This study was designed to elucidate strain- and sex-related differences of carbonyl reductase activity in rat kidney by using the oral antidiabetic drug acetohexamide as substrate. The frequency distribution of carbonyl reductase activities in kidney microsomes of male Fischer 344 (Fischer), Sprague-Dawley, Wistar and Wistar-Imamichi (Wistar-IM) rats exhibited a marked strain-related difference. Furthermore, the enzyme activities in kidney microsomes of Fischer, Sprague-Dawley and Wistar rats were male-specific, resulting insignificant sex-related differences in these strains. There was no sex-related difference of carbonyl reductase activity in kidney microsomes of the Wistar-IM strain, which lacked its activity in both sexes. On the other hand, although carbonyl reductase activities were fully detectable in kidney cytosols from all the strains of male and female rats, no strain- or sex-related difference was observed among the cytosolic enzyme activities. These results provide new information for understanding the influence of internal factors on the renal metabolism of ketone-containing xenobiotics.

In vitro metabolism of LVV-Hemorphin-7 by renal cytosol and purified prolyl endopeptidase

The metabolism of LVVH7, an endogenous peptide obtained by cathepsin D hydrolysis of the β chain of hemoglobin, was studied, in vitro, in the presence of cytosol of rat kidney and compared with angiotensin IV. High metabolic activity was found against these two peptides (half life time <2 min) in this subcellular fraction. The main products of LVVH7 metabolism by renal cytosol are VVH7, H7 and LVVH6 suggesting both aminopeptidase and carboxypeptidase activities. The use of PEP inhibitor in kidney cytosol permitted to demonstrate the major role of prolyl endopeptidase (PEP) in LVVH7 degradation. This fact was reinforced by a kinetic study investigated with purified enzyme ( K m/ V max about 238 mM −1 s −1 and close to that observed for angiotensin related peptides).

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

The B1 Subunit of the H+ATPase Is a PDZ Domain-binding Protein: COLOCALIZATION WITH NHE-RF IN RENAL B-INTERCALATED CELLS

The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype.

Diabetes induces an impairment in the proteolytic activity against oxidized proteins and a heterogeneous effect in nonenzymatic protein modifications in the cytosol of rat liver and kidney.

It is assumed that increased oxidative stress contributes to the development of complications in diabetes. In this study, several markers of protein structural modifications directly induced by free radicals were investigated in the liver and kidney cytosolic fractions of rats with streptozotocin-induced diabetes. Sulfydryl residue and side-chain amino group analyses, as well as immunoblotting and chromatographic measurements of protein-bound carbonyl, suggest that protein oxidative modification is not increased by diabetes, with the exception of sulfydryl groups in renal cytosol. The levels of the glycation-derived carbonyl N epsilon-fructosyl-lysine are significantly increased by diabetes. Furthermore, unchanged proteolytic activity against in vivo-oxidized proteins, significant decreases both in activity against H2O2-modified proteins and in proteasome activity, measured by the degradation of a specific fluorogenic substrate, suggest that the unchanged oxidative protein modification in the diabetic state cannot be attributed to an increased cytosolic proteolytic activity in these tissues. These results provide evidence against a generalized increase in protein oxidative damage and demonstrate a diabetes-induced alteration in cytosolic proteolytic pathways, suggesting that proteasome activity may be impaired in these organs.

Quantitation of α2u-Globulin in Rat Kidney Cytosol by Capillary Electrophoresis

The renal accumulation of α2u-globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat. Several chemicals inducing renal tumors in the male rat were shown to bind to α2u-globulin. This binding impairs the renal degradation of α2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of α2u-globulin accumulation in the renal toxicity of a chemical, a demonstration of the accumulation of this protein in the kidney of the male rat is one prerequisite. Monoclonal antibodies to α2u-globulin are available for quantifying α2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of α2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein–SDS complexes. Using α2u-globulin purified from urine of male rats, the limit of detection was 10 μg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV ≤ 4%) and peak areas corresponding to α2u-globulin (CV ≤ 3%) after normalization to the internal standard was observed. Significant increases in renal α2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with α2u-globulin nephropathy-inducing agents such as trimethylpentane or the alkylphosphonates dimethyl methylphosphonate and diethyl ethylphosphonate, but not in kidney cytosol of male rats treated with tris-(2-chloroethyl)phosphate or the nephrotoxic agent hexachlorobutadiene. A good correlation of the α2u-globulin contents determined by capillary electrophoresis and immunoblotting with an α2u-globulin-specific antibody (r2= 0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of α2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.

Stereoselective formation of glutathione S-conjugates from halovinylmercapturate sulphoxides

1. The glutathione S-transferase catalysed formation of glutathione S-conjugates from halovinylmercapturate sulphoxides was investigated in rat liver and kidney cytosol, with purified glutathione S-transferases and in rat in vivo. 2. The two diastereomers of the sulphoxides of N-acetyl-S-(1,2-dichlorovinyl)-Lcysteine, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine show different reactivities with glutathione and glutathione S-transferases. Rat liver and kidney cytosol catalyses the formation of a 1:1 mixture of two diastereomers of (E)-N-acetyl-S-(2-glutathione-S-yl-2-chlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulphoxide and of (E)-N-acetyl-S-(2-glutathione-S-yl-1,2-dichlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulphoxide. In contrast, only one diastereomer of the Z-isomers was formed. 3. N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulphoxide reacted spontaneously with glutathione at high rates, a 1:1 mixture of both diastereomers of N-acetyl-S-(2-glutathione-S-yl-1-chlorovinyl)-L-cysteine sulphoxide was formed. 4. Metabolism of N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide under by α-class glutathione S-transferases yielded identical products as observed with the cytosolic enzymes. No reaction was observed in the presence of rat liver μ class glutathione S-transferases or human glutathione S-transferase M1. 5. Formation of these glutathione conjugates was also observed in the bile of rat after i.p. administration of the mercapturic acid sulphoxides. The results obtained show that stereochemical aspects may govern the regioselectivity and substrate specificity in glutathione S-transferase-catalysed reactions.

Inhibition of GTPγS-Dependent l-Isoaspartyl Protein Methylation by Tyrosine Kinase Inhibitors in Kidney

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5′- O-[γ-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP. GTPγS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by l-isoaspartyl-containing peptides. GTPγS was the most potent nucleotide tested, whereas GDPβS and ATPγS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein l-isoaspartyl methytransferase (PIMT) activity by GTPγS was reached at a physiological pH in the presence of 10 mM MgCl 2. Other divalent cations, such as Cu 2+, Zn 2+ and Co 2+ (100 μM), totally inhibited GTPγS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPγS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 μM, but increasing the vanadate concentration to more than 40 μM resulted in a dose-dependent inhibition of the GTPγS effect. The tyrosine kinase inhibitors genistein (IC 50= 4 μM) and tyrphostin (IC 50= 1 μM) abolished GTPγS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPγS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPγS-stimulated PIMT activity in the kidney cytosol.

Fate and toxicity of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived mercapturates in male, Fischer 344 rats.

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is formed in the anesthesia circuit by the degradation of sevoflurane. Compound A is nephrotoxic in rats and undergoes metabolism by the mercapturic acid pathway in rats and humans to yield the mercapturates S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L -cysteine (compound 3) and S-[2(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cys teine (compound 5). These experiments were designed to examine the fate and nephrotoxicity of compound A-derived mercapturates in rats. The deacetylation of compounds 3 and 5 by human and rat kidney cytosol and with purified acylases I and III was measured, and their nephrotoxicity was studied in male Fischer 344 rats. The metabolism of the deuterated analogs of compounds 3 and 5, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl ]-N-acetyl-L-cysteine (compound 3-d3) and [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N -acetyl-L-cysteine (compound 5-d3), respectively, was measured. Compound 5, but not compound 3, was hydrolyzed by human and rat kidney cytosols and by acylases I and III. 19F nuclear magnetic resonance spectroscopic analysis showed no urinary metabolites of compound 3, but unchanged compound 5 and its metabolites 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4-carboxylic acid were detected in urine. Compound 5 (250 microM/kg) produced clinical chemical and morphologic evidence of renal injury in two of three animals studied. Compounds 3 and 5 underwent little metabolism. Compound 5, but not compound 3, was mildly nephrotoxic. These results indicate that compound A-derived mercapturate formation constitutes a detoxication pathway for compound A.

Tautomycin inhibits phosphatase-dependent transformation of the rat kidney mineralocorticoid receptor

The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as ‘transformation’. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient ( S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [ 3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4–5.4 S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [ 3H]ALDO-MR complex and the percentage of transformed 5.4 S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form ( K i app =7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.

Native rat kidney mineralocorticoid receptor is a phosphoprotein whose transformation to a DNA-binding form is induced by phosphatases.

Addition of alkaline phosphatase to rat kidney cytosol diminishes the ability of the mineralocorticoid receptor (MR) to bind aldosterone in a time-, temperature- and concentration-dependent form. A variety of phosphatase inhibitors, including levamisole, are effective in preventing this inactivation. On the other hand, when the steroid-receptor complex is incubated in the presence of alkaline phosphatase, an increment in the rate of receptor transformation is evidenced by a change in the sedimentation coefficient from 8.8 S to 5.1 S, as well as increased DNA-binding capacity. The effects of alkaline phosphatase on activation and transformation can also be observed when the MR is incubated at 20 degreesC in the cytosolic medium, indicating that the catalytic action of an endogenous phosphatase may be involved in the transformation process. The ability of phosphatase inhibitors such as levamisole for suppressing both alkaline phosphatase- and endogenous phosphatase-directed transformation does not correspond well between them. Evidence is presented to affirm that the endogenous phosphatase activity is not due to an alkaline phosphatase-type, but it may be due to a protein serine/threonine phosphatase, as evidenced by the inhibitory effects of okadaic acid. The experimental results also show direct evidence that the MR undergoes phosphorylation in a physiological milieu.

Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N-acetyl-L-cysteines.

The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/- 27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.