Purified Rat Kidney Research Articles

A role for divalent metal transporter (DMT1) in mitochondrial uptake of iron and manganese

Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.

Astrocytic localization of kynurenine aminotransferase II in the rat brain visualized by immunocytochemistry

Kynurenic acid (KYNA), a metabolite of the kynurenine pathway of tryptophan degradation, is a neuroinhibitory agent present in the mammalian brain. Endogenous KYNA preferentially affects the alpha7 nicotinic acetylcholine (alpha7nACh) receptor and, possibly, the glycine co-agonist (glycineB) site of the NMDA receptor. Functionally relevant fluctuations in brain KYNA occur under both physiological and pathological conditions, affecting cholinergic and glutamatergic neurotransmission. Kynurenine aminotransferase II (KAT II), the major biosynthetic enzyme of KYNA in the rat brain, catalyzes the irreversible formation of KYNA from its immediate bioprecursor, kynurenine. We now purified rat kidney KAT II to homogeneity, generated a polyclonal rabbit anti-rat KAT II antibody, and purified the antibody using routine biochemical methods. The antibody selectively recognized KAT II by Western blot analysis and in immunotitration experiments. Used for immunocytochemistry, the antibody revealed discrete, specific staining of KAT II-positive astrocyte-like cells throughout the adult rat brain. The presence of KAT II in astrocytes was confirmed by double fluorescence immunostaining with an antibody against the astrocyte-specific marker glial fibrillary acidic protein (GFAP). No specific labeling was detected in neurons or microglia. However, KAT II-positive astrocytes were intimately associated with select neuron populations, supporting a neuromodulatory role of KYNA. Intense staining was frequently seen around brain capillaries, with astrocytic end feet contacting the capillary wall. This may explain the rapid access of blood-derived kynurenine to KAT II-containing astrocytes. The new anti-KAT II antibody should be useful in the further elucidation of the presumed role of KYNA in brain physiology and pathology.

Aminotransferase, l-amino acid oxidase and β-lyase reactions involving l-cysteine S-conjugates found in allium extracts : Relevance to biological activity?

Several cysteine S-conjugates that occur in extracts of garlic and other plants of the allium family possess anti-oxidant properties, and many, including S-allyl- l-cysteine (SAC) and S-allylmercapto- l-cysteine (SAMC), are promising anti-cancer agents. To understand possible biochemical mechanisms contributing to the protective effects, the ability of selected allium-derived l-cysteine S-conjugates to undergo various enzyme-catalyzed transformations was investigated. SAC, SAMC, S-propylmercapto- l-cysteine and S-penta-1,3-dienylmercapto- l-cysteine were shown to be substrates of: (a) highly purified rat kidney glutamine transaminase K (GTK); (b) purified snake venom l-amino acid oxidase; and (c) a cysteine S-conjugate β-lyase present in rat liver cytosol. S-Methylmercapto- l-cysteine was shown to be a substrate of GTK and l-amino acid oxidase, but not of the cysteine S-conjugate β-lyase. Evidence is presented that a major enzyme responsible for the cysteine S-conjugate β-lyase reactions in the rat liver cytosol is γ-cystathionase. The possible role of γ-cystathionase in generating sulfane sulfur from the disulfide-containing cysteine S-conjugates present in allium extracts, and the possible role of this sulfane sulfur in enzyme regulation, targeting of cancer cells and detoxification reactions is discussed. An interesting side finding of the present work is that rat liver mitochondria are more active than rat liver cytosol in catalyzing a cysteine S-conjugate β-lyase reaction with the mitochondrial protoxicant S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) at physiological pH and at low substrate concentration.

Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme–substrate complexes

Background and aims: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. Methods: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0–20 min) and over a wide range of substrate concentrations (0.8–66 μM). Results: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS–PAGE, Western blot and isoelectric focusing (p I 4.9). Its molecular mass is 40±2.3 kDa, determined by SDS–PAGE and 39.8±2 kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis–Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K 1=2.08±0.01 μM and K 2=0.102±0.003 μM. Conclusion: The values of these constants fulfil the restriction 4 K 2≤ K 1 2, necessary for the occurrence of isoenzymes, interpreted in this work as enzyme–substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme–substrate intermediates formed during PBG-D reaction.

Carnitine transport and its inhibition by sulfonylureas in human kidney proximal tubular epithelial cells

The kidney plays an important role in the homeostasis of carnitine by its ability to reabsorb carnitine almost completely from the glomerular filtrate. The transport process responsible for this reabsorption has been investigated thus far only in laboratory animals. Here we report on the characteristics of carnitine uptake in a proximal tubular epithelial cell line derived from human kidney. The uptake process was found to be obligatorily dependent on Na + with no involvement of anions. The process was saturable, with a Michaelis–Menten constant of 14 ± 1 μM. The Na +:carnitine stoichiometry was 1:1. The same process also was found to be responsible for the uptake of acetylcarnitine and propionylcarnitine, two acyl esters of carnitine with potential for therapeutic use in humans. The uptake process was specific for carnitine and its acyl esters. Betaine, a structural analog of carnitine, interacted with the uptake process to a significant extent. The present studies also showed that sulfonylureas, oral hypoglycemic agents currently used in the management of type 2 diabetes, inhibited the carnitine uptake system. Among the sulfonylureas tested, glibenclamide was the most potent inhibitor. The inhibition was competitive. Glibenclamide inhibited the uptake not only of carnitine but also of acetylcarnitine and propionylcarnitine. The inhibition most likely was the result of direct interaction of the compound with the carnitine transporter because the inhibition could be demonstrated in purified rat kidney brush border membrane vesicles.

Acylase-catalyzed deacetylation of haloalkene-derived mercapturates.

Mercapturates (S-substituted N-acetyl-L-cysteines) are terminal metabolites formed by the glutathione-dependent metabolism of electrophilic xenobiotics, including haloalkenes. Acylases catalyze the hydrolysis of N-acyl-L-amino acids, including many xenobiotic-derived mercapturates, to give fatty acids and amino acids as products. Although several acylases have been identified, the acylases that catalyze the deacetylation of the haloalkene-derived mercapturates have not been identified and characterized. Acylase I catalyzes the deacetylation of some haloalkene-derived mercapturates, including S-(1,1,2, 2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine [Uttamsingh, V., et al. (1998) Chem. Res. Toxicol. 11, 800-809]. In the studies presented here, we identified a rat kidney acylase that catalyzed the hydrolysis of the haloalkene-derived mercapturates S-(1, 2-dichlorovinyl)-N-acetyl-L-cysteine, S-(1,2,3,4,4-pentachloro-1, 3-butadienyl)-N-acetyl-L-cysteine, and S-(2,2-dibromo-1, 1-difluoroethyl)-N-acetyl-L-cysteine. The substrate selectivity and amino acid sequence of the purified rat kidney acylase were studied. Although the sequence of the purified rat kidney acylase was somewhat identical with that of aspartoacylase, it did not catalyze the hydrolysis of N-acetyl-L-aspartate.

Molecular Cloning and Functional Characterization of Murine Sphingosine Kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

Purification and Characterization of Rat Kidney Sphingosine Kinase

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9. 27 (mouse). Synaptopodin contains a high amount of proline ( approximately 20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

Phosphorylation of Na,K-ATPase by protein kinase C at Ser18 occurs in intact cells but does not result in direct inhibition of ATP hydrolysis.

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known to be directly phosphorylated on its alpha-subunit, but the functional effects of protein kinases remain controversial. We have developed a sensitive, antibody-based assay for detection of the level of phosphorylation of the alpha1-isoform of rat Na,K-ATPase at the serine residue that is most readily phosphorylated by protein kinase C (PKC) in vitro, Ser18. By stimulation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphorylation (close to 0.9) in several types of intact cells. This demonstrates the accessibility and competency of the site for endogenous phosphorylation. The cells used were derived from rat (NRK 52E, C6, L6, and primary cultures of cerebellar granule cells, representing epithelial cells, glia, muscle cells, and neurons). In the presence of the phosphatase inhibitor calyculin A, full phosphorylation was preserved during subsequent assays of enzyme activity in vitro. Assay of the hydrolysis of ATP in NRK and C6 cells, however, indicated that there was no significant effect of phosphorylation on the Vmax of the Na, K-ATPase or on the apparent affinity for Na+. Any regulatory effect of PKC on sodium pump activity thus must be lost upon disruption or permeabilization of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser18.

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Cloning and Expression of a Rat Kidney Cytosolic Glutamine Transaminase K That Has Strong Sequence Homology to Kynurenine Pyruvate Aminotransferase

An α-keto acid-dependent cysteineS-conjugate β-lyase of rat kidney was identified as cytosolic glutamine transaminase K by Stevenset al.(J. Biol. Chem.261, 15529–15537, 1986). Subsequently, a rat kidney protein with both glutamine transaminase K- and cysteineS-conjugate β-lyase activities was cloned and sequenced by Perryet al.(Mol. Pharmacol.43, 660–665, 1993). This protein was later shown to be identical with kynurenine pyruvate aminotransferase (Moscaet al., FEBS Lett.353, 21–24, 1994). Thus, kynurenine pyruvate aminotransferase possesses both glutamine transaminase K- and cysteineS-conjugate β-lyase-type activities. We have also cloned a cytosolic glutamine transaminase K from a rat kidney cDNA library and expressed the full-length clone in COS1 cells. The transfected cells exhibit marked increases in activities of both glutamine transaminase K and cysteineS-conjugate β-lyase. The enzyme cloned in the present work is a homodimer. Each subunit has a molecular mass of 45.8 kDa and contains 426 amino acid residues. The sequence of cytosolic glutamine transaminase K obtained in the present work has strong similarities to other aminotransferases, including >90% identity with kynurenine pyruvate aminotransferase. Our preparation of purified rat kidney cytosolic glutamine transaminase K possesses some kynurenine pyruvate aminotransferase activity. Thus, rat kidney cytosol possesses at least two distinct enzymes that catalyze glutamine transaminase K/cysteineS-conjugate β-lyase/kynurenine py- ruvate aminotransferase reactions. The present work underscores the difficulties associated with characterizing aminotransferases with overlapping specificities.

Effects of starvation, diabetes and carbon tetrachloride intoxication on rat kidney cortex and liver pyruvate carboxylase levels.

Pyruvate carboxylase (PC) has been quantified in rat liver and kidney cortex under experimental conditions that modify the gluconeogenic response in both organs: fasting, carbon tetrachloride-induced liver degeneration and alloxan-induced diabetes. Enzymatic activity has been assayed by a 14CO2-fixation method. The amount of enzyme has been determined by competitive ELISA using antibodies raised against the purified rat kidney cortex enzyme. Purified fractions of rat-liver and rat-kidney cortex PC have been used as standards. Fasting and carbon tetrachloride administration induced a significant increase (25% to 30%) in the amount of enzyme in liver and kidney cortex. Alloxan-induced diabetes produced a nearly two-fold increase in the hepatic levels of enzyme without a significant modification in the content of the renal enzyme. These results are discussed on the basis of the different metabolic implications of both organs during the physiological or toxic treatments.

Okadaic acid stimulates ouabain-sensitive 86Rb +-uptake and phosphorylation of the Na +/K +-ATPase α-subunit in rat hepatocytes

Ca 2+-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na +/K +-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive 86Rb +-uptake was maximal between two and three minutes and displayed an EC 50 of 41 ± 1 nM. Inhibition of Na +/H + exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na +/K +-ATPase transport. In hepatocytes metabolically-radiolabeled with 32P i, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na +/K +-ATPase α-subunits. In other experiments Na +/K +-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na +/K +-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na +/K +-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 ± 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na +/K +-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na +/K +-ATPase α-subunit.

Demonstration of nuclear translocation of the mineralocorticoid receptor (MR) using an anti-MR antibody and confocal laser scanning microscopy.

Using a synthetic peptide that corresponds to a unique region of the N-terminal domain of the human mineralocorticoid receptor (MR), amino acids 87-96, we have generated a polyclonal antibody, human (h) MRsN. This sequence shares no homology with the corresponding sequences of the glucocorticoid receptor or other steroid/thyroid hormone receptor superfamily members. Antibody hMRsN cross-reacts with MR from human, rat, and mouse cells and recognizes denatured MR from either crude preparations or partially purified rat kidney cytosol, rat colon, or recombinant hMR overexpressed in baculovirus-infected Sf9 cells. Immunoprecipitation of the native MR from either partially purified or crude preparations of rat kidney cytosol with hMRsN, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain, demonstrated a major protein band with a mol wt of 116 kilodaltons. In addition, using confocal laser scanning microscopy and digital image analysis, the immunocytochemical localization of the recombinant hMR over-expressed in Sf9 cells 24 h post-transfection was determined. In the absence of ligand, the MR was detected solely in the cytoplasm, after a 30-min exposure to 100 nM aldosterone the MR was perinuclear, and after 60 min, the MR was predominantly nuclear. To ascertain that this phenomenon was not unique to insect cells, aldosterone induced MR nuclear translocation in mouse macrophage cells was also demonstrated immunocytochemically, clearly indicating a role for nuclear translocation in MR function.

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 m M ethyl-enediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.

Glutamine transaminase K and cysteine S-conjugate β-lyase activity stains

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing l-phenylalanine, α-keto-γ-methiolbutyrate (αKMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and αKMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate β-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing αKMB (to ensure maintenance of the enzyme in the pyridoxal 5′-phosphate form), S-(1,2-dichlorovinyl)- l-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate β-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent M r ∼ 95,000). This band coincides with both the cysteine S-conjugate β-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate β-lyase activity with an R f value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K. Activity staining of rat kidney homogenates (and to a lesser extent rat liver homogenates) for glutamine transaminase K and cysteine S-conjugate β-lyase activities revealed the presence of an additional band. The enzyme responsible for the second band of activity was estimated to have an apparent molecular weight of ∼ 330,000. The ratio of β-lyase to transaminase activity was greater for the higher molecular weight form than for the lower molecular weight form.

Thiopurine methyltransferase regulation in rat kidney: Immunoprecipitation studies

1. Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT activity in the kidneys of male Sprague-Dawley (S-D) rats is approximately twice that present in the kidneys of female S-D rats, and this difference is testosterone-dependent. Renal TPMT activities in these animals also increase dramatically during growth and development. 2. Our studies were conducted to determine whether variations in TMPT activity in the S-D rat kidney were due to differences in the quantity of TPMT protein. Rabbit polyclonal antibodies to partially purified rat kidney TPMT were used to develop an immunoprecipitation assay for immunoreactive TPMT protein. 3. Gender-related differences in renal TPMT activities in S-D rats were due to a lower content of immunoreactive TPMT protein in kidneys of female animals. TPMT enzyme activities and immunoreactive protein levels were also directly correlated in renal preparations from castrated and sham-operated male rats, from testosterone-treated castrated and sham-operated male rats, and from testosterone-treated and control female rats. 4. There was also a significant positive correlation between TPMT enzymic activities and immunoreactive TPMT protein levels in renal tissue from different aged male S-D rats (rs = 0.955, n = 15, P less than 0.001.) 5. These results demonstrate that changes in S-D kidney TPMT activity during growth and development, in the two sexes and in response to testosterone, were due to variations in the quantity of immunoreactive TPMT protein.

Inhibition of aldehyde reductase by aldose reductase inhibitors

A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been examined for their ability to inhibit rat kidney aldehyde reductase (EC 1.1.1.19, EC 1.1.1.20) versus rat lens aldose reductase (EC 1.1.1.21). All aldose reductase. inhibitors examined inhibited aldehyde reductase to some extent both in the reductive reaction as determined with glyceraldehyde as substrate and NADPH as coenzyme, and in the oxidative reaction where l-gulonic acid was oxidized to d-glucuronic acid in the presence of NADP + Of the inhibitors examined, 2,7-dinuorospirofluorene-9,5′-imidazolidine-2′,4′-dione (A11576) was the most potent inhibitor requiring only concentrations in the 10 −8 M range to inhibit 50% of the in vitro activity of rat kidney aldehyde reductase ( ic 50 value), whereas 3-dioxo-1- H-benz[de]isoquinoline-2(3H)-acetic acid (alrestatin) was the least potent inhibitor requiring concentrations in the 10 −5 M range. Both the reductive and oxidative steps appeared equally inhibited by these aldose reductases inhibitors. Moreover, all compounds appeared to inhibit either crude or highly purified rat kidney aldehyde reductase to essentially the same extent. Marked differences in the selectivity of these inhibitors, expressed as the ratio of ic 50 values for rat kidney aldehyde reductase versus rat lens aldose reductase with glyceraldehyde as substrate, were observed with selectivity for aldose reductase ranging from ca. 2-fold for A11576 to 119-fold for 3-(4-bromo-2-nuorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat). Kinetic and competition studies suggest that these inhibitors interact with aldehyde reductase at a common site that is not identical to either the substrate or nucleotide binding site. These results suggest that the inhibitor binding sites of rat kidney aldehyde reductase and aldose reductase contain several common characteristics.

Electrophoretic mobility of γ-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.