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• W2042029123 abstract "A mouse mutant with glutathionuria was discovered by screening for amino acidurias in the progeny of ethylnitrosourea-mutagenized mice. Total glutathione concentration was increased in both blood and urine but decreased in liver homogenates from affected mice. Glutathionuric mice exhibited lethargy, severe growth failure, shortened life spans and infertility. γ-Glutamyl transpeptidase activity was deficient in kidney homogenates of glutathionuric mice. The glutathionuric phenotype in these mice is inherited as an autosomal recessive trait. This mouse mutant will be a useful animal model for the study of γ-glutamyl transpeptidase physiology and glutathione metabolism. A mouse mutant with glutathionuria was discovered by screening for amino acidurias in the progeny of ethylnitrosourea-mutagenized mice. Total glutathione concentration was increased in both blood and urine but decreased in liver homogenates from affected mice. Glutathionuric mice exhibited lethargy, severe growth failure, shortened life spans and infertility. γ-Glutamyl transpeptidase activity was deficient in kidney homogenates of glutathionuric mice. The glutathionuric phenotype in these mice is inherited as an autosomal recessive trait. This mouse mutant will be a useful animal model for the study of γ-glutamyl transpeptidase physiology and glutathione metabolism. GSH is the most abundant cellular thiol and functions as the principal reducing reagent in all cell types (1Meister A. Greenberg D.M. Metabolism of Sulfur Compounds. 3rd Ed. VII. Academic Press, New York1975: 102-188Google Scholar). A partial listing of the antioxidative functions of GSH include: protection against mitochondrial damage, protection against oxygen toxicity in the lung, protection against lipid peroxidation, detoxification of electrophilic compounds through conjugation, preservation of proper sulfide bonds in proteins, a postulated function in anticarcinogenesis, and a role in the immune system (2Larsson A. Orrenius S. Mannervik B. Holmgren A. Functions of Glutathione-Biochemil, Physiological. Raven Press, New York1983Google Scholar). GSH metabolism also provides a source of cysteine for cells (3McIntyre T.M. Curthoys N.P. Int. J. Biochem. 1980; 12: 545-551Crossref PubMed Scopus (84) Google Scholar). γ-Glutamyl transpeptidase (γ-GT; EC2.3.2.2) 1The abbreviations used are: γ-GT, γ-glutamyl transpeptidase; ENU, N-ethyl-N-nitrosourea; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid). catalyzes the initial step in the degradation of GSH (4Curthoys N. Hughey R. Enzyme. 1979; 24: 383-403Crossref PubMed Scopus (133) Google Scholar). γ-GT is a key step in the γ-glutamyl cycle (5Meister A. Tate S.S. Annu. Rev. Biochem. 1976; 45: 559-604Crossref PubMed Scopus (718) Google Scholar), a series of degradative and synthetic reactions that mediate cellular GSH metabolism. Several reviews of γ-GT physiology and function have been published (4Curthoys N. Hughey R. Enzyme. 1979; 24: 383-403Crossref PubMed Scopus (133) Google Scholar, 5Meister A. Tate S.S. Annu. Rev. Biochem. 1976; 45: 559-604Crossref PubMed Scopus (718) Google Scholar, 6Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. I. McGraw-Hill, New York1995: 1461-1477Google Scholar), but despite intensive investigation, the exact role γ-GT plays in GSH metabolism or its putative contribution to renal amino acid transport have not been definitively determined. Bound to secretory endothelial cell membranes in several organs but predominantly in proximal renal tubule cells, γ-GT participates in the transmembrane transport of GSH and in interorgan GSH exchange (Fig. 1) (7Meister A. Fed. Proc. 1984; 53: 3031-3042Google Scholar). Meister (8Meister A. Science. 1973; 180: 33-39Crossref PubMed Scopus (471) Google Scholar) proposed that γ-GT also contributes to amino acid transport in the proximal renal tubule through transpeptidation of GSH and subsequent tubule cell uptake of γ-glutamyl amino acids. In vivomodel systems that have lost γ-GT activity are an exquisitely powerful tool for the study of γ-GT function and its relationship to GSH metabolism. Administration of chemical inhibitors of γ-GT to animals results in both glutathionuria and glutathionemia (9Griffith O.W. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 268-272Crossref PubMed Scopus (206) Google Scholar), but chemically treated animal models are limited by several drawbacks including the temporary nature of inhibition and the difficulty of long-term continuous inhibitor administration. Also, the degree and specificity of enzyme inhibition in various tissues (particularly the brain) of these chemically treated animals is unknown. Genetic γ-GT deficiency has been described in only five humans (6Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. I. McGraw-Hill, New York1995: 1461-1477Google Scholar), and the effects of various different disease states or environmental influences upon γ-GT deficient individuals cannot be adequately evaluated given the rarity of the disorder. A genetic animal model of total γ-GT deficiency overcomes the limitations of previously existing experimental systems and provides a useful tool for the study of γ-GT function and GSH physiology. N-Ethyl-N-nitrosourea (ENU) is the most potent point mutagen known (10Russell W.L. Kelly E.M. Hunsicker P.R. Bangham J.W. Maddux S.C. Phipps E.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5818-5819Crossref PubMed Scopus (445) Google Scholar), yielding mutation rates up to 300 × 10−5 in mice, depending upon the specific locus tested. ENU mutagenesis has been used by several laboratories to generate mutant mouse strains that model specific human genetic diseases (11Harding C.O. McDonald J.D. Wolff J.A. Monastersky G.M. Robl J.M. Strategies in Transgenic Animal Science. ASM Press, Washington, D. C.1995: 245-270Google Scholar). Since the report of phenylketonuria secondary to phenylalanine hydroxylase deficiency in ENU-generated mouse mutants (12Shedlovsky A. McDonald J.D. Symula D. Dove W.F. Genetics. 1993; 134: 1205-1210Crossref PubMed Google Scholar), our laboratory has focused upon developing mouse models of other human inborn errors of metabolism. To this end, we screen the progeny of ENU-treated mice for metabolic abnormalities using a variety of urine biochemical analyses. Using this protocol, we have previously isolated a mouse strain with recessively inherited sarcosine dehydrogenase deficiency (13Harding C.O. Williams P. Pflanzer D.M. Colwell R.E. Lyne P.W. Wolff J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2644-2648Crossref PubMed Scopus (23) Google Scholar). In this report, we describe a genetic mouse model of autosomal recessively inherited γ-GT deficiency developed by random mutagenesis using ENU. These mice exhibit glutathionuria/emia, severe growth failure, shortened life spans, and inability to reproduce. This strain, designated GGT enu1, provides a useful experimental system for the study of γ-GT physiology and GSH metabolism. All reagents were purchased from Sigma unless otherwise specified. Male C57Bl/6J mice were treated with 50 mg/kg body weight ENU weekly for a total of three doses according to the method of King et al. (14King T.R. Dove W.F. Herrmann B. Moser A.R. Shedlovsky A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 222-226Crossref PubMed Scopus (29) Google Scholar). Breeding and metabolic screening of potentially mutant progeny was carried out as described previously (13Harding C.O. Williams P. Pflanzer D.M. Colwell R.E. Lyne P.W. Wolff J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2644-2648Crossref PubMed Scopus (23) Google Scholar). Urine samples were collected on filter paper from all G3 progeny after weaning (21–28 days of age) following an overnight fast. Each urine sample was evaluated using a battery of qualitative chemical analyses including sodium cyanide-sodium nitroprusside reagent for the detection of disulfides and free sulfhydryls (15Shih V.E. Mandell R. Sheinhait I. Hommes F. Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 49-68Google Scholar). Urine amino acids were examined with one-dimensional paper chromatography using butanol:acetic acid:water (12:3:5) solvent and detection with ninhydrin according to the method of Smith (16Smith I. Chromatographic and Electrophoretic Techniques. Interscience, New York1960: 82-142Google Scholar). Quantitative urine and plasma amino acid analysis and quantitative urinary organic acid analysis were performed in any mouse that had an abnormal result by amino acid paper chromatography. Quantitative urine and plasma amino acids were analyzed on a Beckman System 6300 automatic amino acid analyzer according to the methods of Slocum and Cummings (17Slocum R.H. Cummings J.G. Hommes F.A. Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual. Wiley-Liss, New York1991: 87-126Google Scholar). Quantitative urine organic acids were measured by trimethylsilyl derivatization followed by gas chromatography-mass spectrometry according to the method of Hoffmannet al. (18Hoffmann G. Aramaki S. Blum-Hoffmann E. Nyhan W.L. Sweetman L. Clin. Chem. 1989; 35: 587-595Crossref PubMed Scopus (170) Google Scholar). Duplicate one-dimensional paper chromatograms were examined specifically for the presence of sulfur-containing amino acids using platinic iodide reagent (15Shih V.E. Mandell R. Sheinhait I. Hommes F. Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 49-68Google Scholar) in any mouse that had an abnormal cyanide-nitroprusside screening test. Urine from the mouse mutant described in this report and the identity of the sulfur-containing compounds therein were further examined by two-dimensional high voltage thin layer chromatography with ninhydrin detection (15Shih V.E. Mandell R. Sheinhait I. Hommes F. Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 49-68Google Scholar) in the laboratory of Dr. Vivian Shih, Massachusetts General Hospital, Boston, MA. Total glutathione concentration in urine, plasma, and tissues of control and glutathionuric (GGT enu1) mice was determined enzymatically using the method of Tietze (19Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5556) Google Scholar). GSH reacts with the sulfhydryl reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB or Ellman's reagent) to produce 2-nitro-5-thiobenzoic acid and its glutathionyl adduct. Yeast glutathione reductase added to the reaction mixture reduces GSSG in the presence of NADPH to form GSH which is then free to react with DTNB. The rate of 2-nitro-5-thiobenzoic acid production is measured by monitoring absorbance at 412 nm and is proportional to the total GSH concentration in the solution. For the assay, all reagents are prepared in 125 mm sodium phosphate, pH 7.5, 6.3 mm sodium EDTA buffer. 700 μl of 0.3 mm NADPH, 100 μl of 6 mm DTNB, and 200 μl of sample are combined in a 1-ml cuvette and allowed to equilibrate at 30 °C in a water bath for 5 min. The reaction was initiated by adding 10 μl of 50 unit/ml yeast glutathione reductase, and the increase in absorbance at 412 nm was recorded for 6 min at 30 °C. Total glutathione concentration in unknowns was calculated from a standard curve of known GSSG concentrations varying from 0 to 0.02 mm but are reported as millimoles/liter GSH. The absorbance at 412 nm does not appreciably change over 6 min without the addition of glutathione reductase. Urine samples for total glutathione measurement were collected from urine- soaked filter paper by NH4OH elution. Urine glutathione concentration was measured without further sample modification and was corrected for urine creatinine concentration. Blood was obtained by direct cardiac puncture and anticoagulated with 25 μl of 0.1 m EDTA. Plasma, collected by centrifugation, was deproteinized with 0.1 volume 35% w/v sulfosalicylic acid. Following centrifugation, total glutathione concentration was measured in the supernatant. Solid tissue samples were weighed and homogenized in 10 volumes of 10% w/v trichloroacetic acid with five up and down strokes of a Pro2000 tissue homogenizer (Pro Scientific, Inc.). Following centrifugation at 3000 × g, total glutathione concentration was measured in the supernatant and corrected for wet weight of the tissue sample. γ-GT activities in whole kidney homogenates of control (C57Bl/6J) and GGT enu1 mice were determined by the method of Orlowski and Meister (20Orlowski M. Meister A. Biochim. Biophys. Acta. 1963; 73: 679-681Crossref PubMed Google Scholar). 10% w/v kidney homogenates were prepared in 0.1 m Tris-HCl, pH 9.0, with five strokes of a Pro2000 electric homogenizer while the samples remained on ice. The samples were kept on ice or stored at −20 °C until γ-GT activity was measured. In this assay, the γ-glutamyl moiety of the artificial substrate, γ-glutamyl-p-nitroanilide, is transferred to glycylglycine by γ-GT and the optically active product, p-nitroaniline, is generated. 1.8 ml of 11.11 mm glycine-glycine + 2.78 mm γ-glutamyl-p-nitroanilide in 0.1m Tris-HCl, pH 9.0, is added to 10–500 μg of sample protein in a 200-μl volume. The reaction is incubated at 37 °C for 10 min and then stopped with the addition of 50 μl of 50% w/v trichloroacetic acid followed by 2 ml of 2 m acetic acid. The absorbance of the sample at 410 nm is measured versus a duplicate reaction to which trichloroacetic acid and acetic acid had been added immediately to stop the reaction at time 0. The amount ofp-nitroaniline produced in the reaction was calculated using the molar absorptivity of p-nitroaniline at 410 nm (ε = 8.8 mm−1 cm−1) and γ-GT activity was expressed in terms of nanomoles ofp-nitroaniline produced/min/mg of protein. Protein concentrations were determined by a modification of a bicinchoninic acid method (Pierce) (21Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18712) Google Scholar). γ-GT was partially purified from wild type Harlan Sprague Dawley rat, wild type C57Bl/6J mouse, andGGT enu1 mouse kidney homogenates according to the method of Hughey and Curthoys (22Hughey R.P. Curthoys N.P. J. Biol. Chem. 1976; 251: 7863-7870Abstract Full Text PDF Google Scholar). Briefly, kidney microsomal fractions were isolated by differential centrifugation and resuspended in 0.1 m Tris buffer, pH 9.0, 1% Triton X-100. Alkaline phosphatase (Sigma diagnostic kit) and γ-GT activities were measured on the microsomal fractions from each animal. Protein electrophoresis was performed on SDS-10% polyacrylamide gels (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar), and proteins were transferred by electrophoretic elution (24Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar) to polyvinylidene difluoride membranes (Immobilon-P, Millipore). γ-GT protein was identified using polyclonal rabbit antisera (courtesy of Dr. Henry Pitot) raised against purified rat γ-GT. As a control, an integral renal tubule membrane protein, the β1 subunit of Na,K-ATPase, was detected on a duplicate immunoblot using polyclonal rabbit sera raised against amino acids 152–340 of the rat Na,K-ATPase β1 subunit as deduced from the cDNA sequence (Upstate Biotechnology, Lake Placid, NY). On both blots, the primary antibodies were localized with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma), and alkaline phosphatase activity was detected using nitro blue tetrazolium/bromochloroindolyl phosphate substrate (Pierce). Male C57Bl/6J inbred mice were treated weekly with ENU by intraperitoneal injection for a total of three doses as described previously (14King T.R. Dove W.F. Herrmann B. Moser A.R. Shedlovsky A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 222-226Crossref PubMed Scopus (29) Google Scholar). After the mice had regained fertility, each was bred to C57Bl/6J females to generate five to ten G1 males. G1 (n = 31) males were bred with females to generate G2 offspring and then each of the G1 males were back-crossed with two or three of their G2 daughters to generate a total of 553 potentially homozygous mutant G3 mice. Urine samples from two male mice in a single sibship gave a positive test result with sodium cyanide-sodium nitroprusside reagent, indicating the presence of a disulfide or free sulfhydryl-containing compound in the urine. Qualitative urine amino acid analysis by one-dimensional paper chromatography and ninhydrin detection demonstrated a large red-purple spot with R F 0.13 and a smaller pink spot withR F 0.31 which are not present on the amino acid chromatograms of control mouse urine. The larger spot reacted with both the cyanide-nitroprusside and iodoplatinate reagents indicating the presence of sulfur in the compound. This large spot had the sameR F and color on ninhydrin-staining as GSSG purchased from Sigma. The smaller pink spot corresponded to GSH, which was present at much lower concentrations than GSSG in the mutant mouse urine. Oxidized and reduced glutathione were also identified by two-dimensional high voltage electrophoresis of mutant mouse urine by Drs. Kimiyo Mogami and Vivian Shih, Massachusetts General Hospital, Boston, MA. Quantitative urine amino acid analysis using the Beckman amino acid analyzer (high pressure liquid chromatography) showed three abnormal peaks on the chromatogram (Fig. 2). A single, sharp peak with a retention time of 12.0 min co-chromatographed with both GSH and γ-glutamylcysteine. A broad peak at 18–27 min corresponded to GSSG. The third peak at 37 min was broad and smaller than the first two peaks and has been tentatively identified as bis-γ-glutamylcystine (γ-glutamylcysteine disulfide), a substance which has also been detected in the urine of humans with γ-GT deficiency (25Griffith O.W. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3384-3387Crossref PubMed Scopus (105) Google Scholar). The method of urine collection used in our screening protocol likely promoted oxidation of GSH to GSSG (and of γ-glutamylcysteine to bis-γ-glutamylcystine), yielding the large amount of GSSG compared with GSH in most urine samples from the affected mice. Pretreatment of mutant mouse urine with dithiothreitol resulted in complete disappearance of the broad peaks associated with GSSG and bis-γ-glutamylcystine and accentuation of the peak corresponding to GSH and γ-glutamylcysteine (data not shown). At birth, glutathionuric mice were indistinguishable from their unaffected litter mates. The total number of offspring in litters which contained glutathionuric pups was not significantly different from the size of control C57Bl/6J litters (5–12 pups/litter) in our colony. By approximately 14 days of age, glutathionuric pups appeared smaller than their litter mates, and at weaning (3–4 weeks of age), the body weight of affected mice was significantly lower (Fig. 3). Other than their size, the mice at weaning appeared physically healthy and morphologically normal (Fig. 4).Figure 4Physical phenotype of GGT mice. A male glutathionuric GGT enu1 mouse (left) and a phenotypically normal male sibling (right) at 5 weeks of age.View Large Image Figure ViewerDownload (PPT) Severe growth failure persisted after weaning (Fig. 3). As the glutathionuric mice aged, their fur became rough and dull, but there was no change in coat color. The mice became less active, but were easily agitated. Many of the glutathionuric mice developed a severe thoracolumbar kyphosis. One severely kyphotic mouse developed paralysis of the hindlimbs, and two males suffered maceration of the penis due to priapism. Approximately 10% of the mice exhibit unilateral cataracts which are physically apparent by 2–3 months of age. A single mouse had bilateral cataracts. Unfortunately, neither male nor female glutathionuric mice have produced offspring. The line of glutathionuric mice has been maintained with considerable difficulty only by breeding unaffected siblings of glutathionuric mice which potentially are heterozygous for the glutathionuric trait. Gross anatomic examination of several mice revealed no abnormalities except for small size of all organs in proportion to the small body size overall. In one mouse sacrificed at age 14 months, a large cystic mass completely replaced the right kidney. The right ureter, urinary bladder, and the left kidney and ureter appeared grossly normal. In another mouse sacrificed at age 15 months, a solitary solid tumor was found in the liver. Aortic perfusion with formalin-saline was performed on two male glutathionuric mice. Microscopic examination of most internal organs with hematoxylin-eosin staining was completely normal except for oligospermia apparent in the testes. Nissl staining of coronal sections from the lens of a single mouse confirmed the subcapsular location of a clinically apparent unilateral cataract. Histology of the contralateral lens in that mouse was normal. The brain and spinal cord of two mice were sectioned and examined with hematoxylin-eosin or Nissl staining. No anatomic abnormalities of the nervous system were detected. The hematocrit of four different glutathionuric mice was similar to that of control mice, ranging from 50–55%. The microscopic appearance of Wright-stained peripheral blood smears from glutathionuric mice was normal compared with blood smears from control C57Bl/6J mice. Neither male nor female glutathionuric mice have successfully produced offspring. The mean age of glutathionuric mice (n = 60) at death was 242 ± 15 days (±S.E.) with a range of 70–512 days, while the phenotypically normal siblings of glutathionuric mice (n = 88) lived an average of 339 ± 22 days (p < 0.001) with a range of 74–929 days. In addition to the large amount of glutathione detected, quantitative urine amino acid analysis revealed slight elevations of many amino acids including threonine, glycine, cystine, isoleucine, leucine, ornithine, and lysine compared with control mouse urine samples (Table I). This may indicate a mild generalized defect of amino acid reabsorption in the renal tubules of glutathionuric mice. Additionally, taurine, a sulfur-containing amino acid that is usually the most abundant amino acid in control mouse urine, was consistently low in urine from glutathionuric mice. Plasma cystathionine levels were slightly lower in mutant mice (3.1 ± 3.8 μmol/liter) than in controls (8.5 ± 1.8 μmol/liter), but no other significant differences in plasma amino acid concentrations were detected. Specifically, the plasma levels of other sulfur-containing amino acids including methionine and non-protein-bound cystine were normal in glutathionuric mice. Quantitative amino acid analysis was performed on trichloroacetic acid extracts of liver, kidney, and brain of glutathionuric and control mice. No significant differences in any amino acids including free cystine were detected (data not shown).Table IQuantitative urine amino acid analysisAmino acidGGT enu1 (n = 5)1-aKey: *p < 0.05; **p< 0.01; xxxp < 0.001 by Student's ttest.Wild type (n = 12)aKey: *p < 0.05; **p< 0.01; xxxp < 0.001 by Student's ttest.mmol/g creatinineTaurine3.41 ± 1.99*7.16 ± 2.79Threonine0.464 ± 0.194xxx0.169 ± 0.0616Glycine1.97 ± 1.08**0.886 ± 0.344Cystine0.488 ± 0.385**0.172 ± 0.0439Methionine0.127 ± 0.1180.153 ± 0.0612Cystathionine0.161 ± 0.1480.0969 ± 0.0709Isoleucine0.752 ± 0.181xxx0.151 ± 0.121Leucine0.383 ± 0.173*0.217 ± 0.117Ethanolamine0.330 ± 0.120**1.44 ± 0.725Ornithine1.51 ± 1.30*0.565 ± 0.317Lysine0.253 ± 0.131xxx0.0896 ± 0.0424Amino acid concentrations in urine of glutathionuric (GGT enu1) and wild type (C57B1/6J) mice corrected for urine creatinine concentration. Values expressed as mean concentration ± 1 S.D. Only data for sulfur-containing amino acids and those amino acids which were significantly elevated inGGT enu1 mice are shown.a Key: *p < 0.05; **p< 0.01; xxxp < 0.001 by Student's ttest. Open table in a new tab Amino acid concentrations in urine of glutathionuric (GGT enu1) and wild type (C57B1/6J) mice corrected for urine creatinine concentration. Values expressed as mean concentration ± 1 S.D. Only data for sulfur-containing amino acids and those amino acids which were significantly elevated inGGT enu1 mice are shown. Using the method of Tietze (19Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5556) Google Scholar), glutathione concentration was measured in urine and plasma, and trichloroacetic acid extracts of liver, kidney, and brain from glutathionuric and control mice (Table II). As expected, urine glutathione excretion was very elevated in the mutant mice. Plasma glutathione concentration was also elevated in the mutant mice. Total glutathione was also elevated in trichloroacetic acid extracts of kidney and brain from glutathionuric mice. However, total glutathione in liver extract from glutathionuric mice was significantly decreased compared with control mice.Table IITissue total glutathione concentrationsTissueTotal glutathioneaData expressed as mean concentration ± 1 S.D. **p < 0.01; xxxp < 0.001 by Student's t test.GGTenu1ControlUrine (μmol GSH/mg creatinine)68.8 ± 32.3xxx0.116 ± 0.157(4 mice, 10 samples)(8 mice, 8 samples)Plasma (μmol GSH/liter)213 ± 125xxx42.5 ± 18.0(5 mice, 13 samples)(4 mice, 5 samples)Kidney (μmol GSH/g tissue wet weight)3.27 ± 1.98xxx1.21 ± 1.38(5 mice, 17 samples)(9 mice, 26 samples)Liver (μmol GSH/g tissue wet weight)3.07 ± 1.73xxx8.93 ± 3.30(5 mice, 14 samples)(11 mice, 37 samples)Total glutathione (GSH + GSSG) concentrations in tissues from glutathionuric (GGT enu1) and control (C57B1/6J) mice.a Data expressed as mean concentration ± 1 S.D. **p < 0.01; xxxp < 0.001 by Student's t test. Open table in a new tab Total glutathione (GSH + GSSG) concentrations in tissues from glutathionuric (GGT enu1) and control (C57B1/6J) mice. γ-GT-specific activities were measured in Tris-HCl extracts of whole kidney from glutathionuric and control mice (Table III). Kidney γ-GT activity of glutathionuric mice was nearly 100-fold lower than γ-GT activity measured in control C57Bl/6J mice. Under reaction conditions similar to those used to measure γ-GT activity in kidney extracts from control mice (25–50 μg of protein added to the reaction and 10 min of incubation), γ-GT activity was frequently undetectable in kidney extracts from glutathionuric mice. Increasing the amount of glutathionuric mouse kidney extract in the reaction to 500 μg of protein allowed the measurement of a slight amount of γ-GT activity. Incubation of the reaction with glutathionuric mouse kidney extract at 37 °C for up to 4 h did not result in appreciably higherp-nitroaniline production (data not shown). A 1:1 mixture of kidney extracts from a glutathionuric mouse and from a control mouse had measured γ-GT activity equaling 50% of specific γ-GT activity measured in control extracts alone, indicating that kidney extract from glutathionuric mice did not inhibit the γ-GT reaction when active enzyme was added. Kidney γ-GT activity was also measured in four mice that had produced glutathionuric offspring and were therefore carriers of the glutathionuric trait. Mean kidney γ-GT activity in the carriers was 72% of that in control mice.Table IIIKidney γ-glutamyl transpeptidase activityGGT enu1GGT enu1/+Controlnmol p-nitroaniline produced/mg protein/min10.2 ± 19.8*aData expressed as mean ± 1 S.D. *p < 0.001, **p < 0.02 by Student'st test.573 ± 239**789 ± 327(n = 14, 4 mice)(n = 14, 4 mice)(n = 43, 12 mice)γ-Glutamyl transpeptidase activity in kidney extracts from glutathionuric (GGT enu1), heterozygous (GGT enu1/+) and control (C57B1/6J) mice.a Data expressed as mean ± 1 S.D. *p < 0.001, **p < 0.02 by Student'st test. Open table in a new tab γ-Glutamyl transpeptidase activity in kidney extracts from glutathionuric (GGT enu1), heterozygous (GGT enu1/+) and control (C57B1/6J) mice. Kidney microsomal fractions from the Harlan Sprague Dawley rat, C57Bl/6J mouse, and GGT enu1mouse were analyzed for the presence of γ-GT protein by Western blot analysis using polyclonal rabbit sera raised against purified rat γ-GT protein. The specific activity of γ-GT in each kidney microsomal fraction was 557 ± 10.1 nmol/min/mg of protein in wild type C57Bl/6J mouse, 64.6 ± 5.9 nmol/min/mg inGGT enu1 mouse, and 2070 ± 365 nmol/min/mg in the Harlan Sprague Dawley rat. The specific activity of alkaline phosphatase, another renal tubule membrane-associated enzyme, in these microsomal fractions was 788 ± 39.1 nmol/min/mg of protein in the wild type C57Bl/6J mouse (ratio of γ-GT to alkaline phosphatase activity = 0.71), 839 ± 35.2 nmol/min/mg in theGGT enu1 mouse (ratio = 0.071), and 703 ± 16.6 nmol/min/mg in the Harlan Sprague Dawley rat (ratio = 2.9). Aliquots containing approximately 10 μg of total protein from each microsomal fraction were loaded onto a 10% SDS-polyacrylamide gel. Immunoblotting revealed two densely stained bands with apparent molecular masses of approximately 55 and 35 kDa in the rat kidney microsomal fraction (Fig. 5). This result agrees with the immunoblot of purified rat γ-GT obtained by Coloma and Pitot (26Coloma J. Pitot H.C. Biochem. Biophys. Res. Commun. 1986; 135: 304-308Crossref PubMed Scopus (7) Google Scholar). Two additional less intensely stained bands with apparent molecular masses of 150 and 25 kDa were also detected in the rat. The 150-" @default.
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• W2042029123 date "1997-05-01" @default.
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• W2042029123 title "Mice with Genetic γ-Glutamyl Transpeptidase Deficiency Exhibit Glutathionuria, Severe Growth Failure, Reduced Life Spans, and Infertility" @default.
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