A novel inhibitor of beta-glucuronidase: L-aspartic acid.

Infants who consume casein hydrolysate formula have been shown to have lower neonatal jaundice levels than infants who consume routine formula or breast milk. Because casein hydrolysate has been shown to contain a beta-glucuronidase inhibitor, one possible mechanism to explain this finding is blockage of the enterohepatic circulation of bilirubin by a component of the formula. The aim of this research was to identify the source of the beta-glucuronidase inhibition in hydrolyzed casein. A beta-glucuronidase inhibition assay and measurements of physical and kinetic parameters were used to analyze the components of hydrolyzed casein and infant formulas. Kinetic studies used purified beta-glucuronidase. The L-aspartic acid in hydrolyzed casein accounts for the majority of the beta-glucuronidase inhibition present. Kinetic studies indicate a competitive inhibition mechanism. L-aspartic acid is a newly identified competitive inhibitor of beta-glucuronidase.

Increased immunoreactivity of glutathione-S-transferase in the retina of Swiss Webster mice following inhalation of JP8 + 100 aerosol.

The current study was designed to determine whether exposure of mice to aerosolized jet fuel (JP8 + 100) resulted in changes in the cellular distribution or immunoreactivity of the enzyme glutathione S-transferase (GST), a biomarker of toxicant exposure. Male mice were exposed to JP8 + 100 at 1000 mg/m3 or 2500 mg/m3 in aerosol for 1 h per day for 7 days and then sacrificed. The retinas were studied by immunohistochemical methods. The JP8 + 100 exposure caused a marked increase in the immunoreactivity of anti-GSTM antibodies with the radial glial cells of the retina, the Müller cells. These results are consistent with the hypothesis that JP8 + 100 acts as a toxicant to mouse retina by permitting the flux of materials across the blood-retina barrier. The findings are relevant to humans because recent studies indicate that Air Force personnel assigned to clean and maintain fuel pods may be exposed to concentrations of JP8 + 100 exceeding 1000 mg/m3.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Maturation of cochlear glutathione-S-transferases correlates with the end of the sensitive period for ototoxicity.

The developing mammalian cochlea is especially sensitive to chemical toxins. In rats, the period of increased sensitivity falls roughly between postnatal days (P) 8 and 28. One unexplored hypothesis for this 'sensitive period' is that young cochleas may have immature complements of detoxification enzymes. Glutathione-S-transferases (GSTs) are a family of detoxification enzymes which catalyze the conjugation of many xenobiotics to glutathione. Using high performance liquid chromatography (HPLC), we measured the concentrations of soluble GST isoforms in cochleas of developing Fischer 344 rats. At P1, the concentration of isoform rGSTP1 was 9 pmol/mg protein. That of the remaining isoforms studied was low, <2 pmol/mg protein, and, except for rGSTA3, remained so throughout the period of study. At P2, immunolabelling visualized rGSTP1 in the stria vascularis, Reissner's membrane, spiral limbus and organ of Corti. From P1 to P28, rGSTP1 increased to 15 pmol/mg protein and was detected additionally in satellite cells of the spiral ganglion and in the spiral ligament. From P7 to P28, rGSTA3 increased 8-fold (3-24 pmol/mg protein), became the predominant isoform in the adult organ and localized to pillar cells, the limbus and the spiral ligament. In the vestibule, rGSTP1 predominated, although rGSTA3 increased slightly over time. These observations suggest that biochemical immaturity in detoxification enzymes in the cochlea may contribute to the increased sensitivity to ototoxins during development and that differences in detoxification enzymes between cells in the cochlea and between inner ear organs may underlie differences in susceptibility to ototoxins.

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro- and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead-induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of lead's renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two-dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either or) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix-assisted laser desorption/ionization mass spectrometry/electrospray ionization-mass spectrometry (MALDI-MS/ESI-MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of lead's nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Differential expression of cytosolic proteins in the rat kidney cortex and medulla: preliminary proteomics.

The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.

Effects of lead on rat kidney and liver: GST expression and oxidative stress.

The effect of acute exposure to lead acetate on the expression of glutathione S-transferase (GST) subunits and the levels of reduced and oxidized glutathione (GSH) and malondialdehyde (MDA) in rat kidney and liver was determined. The purpose of this study was to determine if GSH depletion and/or oxidative stress were responsible for changes in the expression of some or all GSTs that followed lead exposure. In kidney, all GST subunits increased following injection of lead. The level of kidney GSH was not changed at either 0.5 or 1 h after lead exposure, but increased 3, 6, 12 and 24 h after a single injection of lead. MDA levels (a marker of lipid peroxidation) did not change in kidney following lead injection. Immunohistochemical markers of oxidative stress and nitric oxide production were also unchanged by lead administration. Therefore. we conclude that the increases in GST levels in kidney following lead exposure were not dependent on oxidative stress. In liver, lead injection caused GSH depletion (61% of control 12 h after lead treatment) and increased MDA production (2.5-fold increase 6 h after lead exposure), while GSTA1, GSTA2, GSTM1 and GSTM2 did not increase. Analysis of the effects of lead on GST mRNA and GST cellular localization were performed by Northern blot and immunohistochemical techniques. Immunoperoxidase light microscopy and immunogold electron microscopy revealed that the increase in kidney GSTM1 and GSTP1 occurred in nuclei, cytoplasm and microvilli of proximal tubules. Northern blot analysis of GSTA2 and GSTP1 mRNAs showed that their increase following lead exposure was inhibited by actinomycin D, suggesting transcriptional induction. This study demonstrates that acute lead exposure causes dramatic changes in the subcellular distribution and expression of rat kidney GSTs, and that these changes are not a result of oxidative stress.

Glutathione S-transferases: two-dimensional electrophoretic protein markers of lead exposure.

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

Effects of lead on glutathione S-transferase expression in rat kidney: a dose-response study.

Glutathione S-transferases (GST, EC 2.5.1.18) are a family of phase II detoxification enzymes involved in the conjugation of glutathione to a highly diverse group of compounds. The purpose of this study was to evaluate the dose-response effects of lead acetate administration on the expression of rat kidney GST. Sprague-Dawley rats were injected with doses of lead acetate ranging from 0.11 to 114 mg/kg (0.3 to 300 mumol/kg) for three consecutive days and sacrificed 24 h later. Kidney GST activity, GST isoform HPLC profiles, blood lead analysis, and electron microscopy were performed. A dose of 1.1 mg/kg lead acetate resulted in a blood lead level of 26 micrograms/dl and produced a significant increase in GST activity which continued to increase with dose up to 38 mg/kg. Morphological changes were detected at 3.8 mg/kg and increasing severity of cellular damage paralleled dose, blood lead levels, and changes in body weight. Individual GST isoforms exhibited different thresholds and maxima; rGSTP1 and rGSTM1 had thresholds of 1.1 and 3.8 mg/kg, respectively, very similar rates of increase with dose, and a maximum yield that was 450% above control at a dose of 38 mg/kg for both enzymes. rGSTA1 and rGSTA3 showed similar thresholds (1.1 mg/kg) and maximal fold increase (275%) but varied in the relative response to each dose. These results indicate that renal GST increases occur at lead levels which are environmentally significant, that these changes precede cellular damage, and suggest that GST may serve as a tissue biomarker of lead exposure.

Effects of triethyl lead administration on the expression of glutathione S-transferase isoenzymes and quinone reductase in rat kidney and liver.

The effects of triethyl lead chloride (TEL) on the expression of two detoxication enzyme families, glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (QR) were determined in rat liver and kidney. Fischer 344 rats were given one intraperitoneal (i.p.) injection of TEL. GST activity, GST isoenzyme levels, mRNA levels of alpha class GST isoenzymes Ya1, Ya2, and Yc1 and activity of QR were determined. Treatment of rats with TEL caused a significant increase in GST activity in kidney. In kidney, the levels of all GST subunits were significantly elevated; the largest increase was a 3.2-fold increase in GST Yb1. The levels of GST Ya1, Ya2, and Yc1 mRNA also increased after injection of TEL. In liver, TEL injection resulted in decreased GST activity and lower levels of hepatic GSTs Yb2, Yb3, Ya1, and Ya2. The largest decrease was a 40% reduction of GST Ya1. In contrast, the level of liver GST Yc1 increased from day 4 through day 14 after injection of 10 mg/kg TEL and Yp was increased 1.4-fold 4 days after injection of 12 mg/kg TEL. The levels of liver mRNAs coding for alpha class GSTs Ya1, Ya2, and Yc1 were reduced 12 h after injection of TEL. The mRNA levels of GST Ya1 and Ya2 returned to basal level while Yc1 message increased to a level higher than controls 24 h after TEL injection. The increase in Yc1 protein between days 4 and 14 is consistent with the increase in the corresponding mRNA. The activity of QR was elevated 1.5-fold in kidney and 2.7-fold in liver 14 days after the injection of TEL. This report demonstrates that administration of organic lead significantly affects GST expression and QR activity in a tissue-specific and isoenzyme-specific manner. These results indicate that GST expression and QR activity are not co-regulated.

Increased levels of glutathione S transferases and appearance of novel alpha class isoenzymes in kidneys of mice exposed to mercuric chloride. I. Biochemical and immunohistochemical studies.

Glutathione S transferases (GST) are a family of enzymes that detoxify electrophilic xenobiotics. This enzyme family was examined in kidneys of mice exposed to mercuric chloride, a known nephrotoxin, because GST have been shown to protect cells against toxicant-induced damage and may serve as biomarkers for toxicant exposure. The purpose of this study was to determine the effect of mercuric chloride on GST activity, isoenzyme levels, and cellular localization in the kidney of Swiss Webster mice. The cellular localizations of alpha, mu, and pi class GST in the kidneys of control and mercuric chloride treated mice were studied immunohistochemically. The GST isoenzyme levels were measured by high-performance liquid chromatography. The mice treated with mercuric chloride had (1) increased amounts of GSTA1/A2 protein in kidney homogenates as compared with controls when analyzed by chromatography and electrophoresis; (2) two new isoforms of the alpha isoenzyme in kidney as demonstrated by Western blot, polyacrylamide gel electrophoresis, and high-performance liquid chromatography, and (3) increased reactivity between antibodies, against GSTA1/A2 or GSTM1 isoenzymes, and cells in the proximal and distal renal tubules as shown by immunohistochemical techniques. The authors conclude that the GSTA1/A2 may protect those cells in the proximal and distal tubules of the renal cortex from toxicant effects of mercuric chloride. This would be one general mechanism for cell protection against a wide variety of toxicants including heavy metals and halogenated aromatics.

Calmodulin N-methyltransferase. Kinetics, mechanism, and inhibitors.

The present study was undertaken to determine kinetic and inhibition parameters and the mechanism of S-adenosyl-L-methionine:calmodulin-L-lysine N6-methyltransferase (EC 2.1.1.60, CLNMT), an enzyme for which calmodulin is a substrate. Partially purified CLNMT isolated from rat testes had a Vmax of 540 pmol/min/mg and Km values for mushroom demethylcalmodulin and S-adenosyl-L-methionine of 230 nM and 2.0 microM, respectively. Kinetic analysis indicated a complex Bi Bi sequential kinetic mechanism for CLNMT where S-adenosyl-L-methionine binds initially and is followed by demethylcalmodulin binding. When the effects of 20 different compounds that are either inhibitors of calmodulin-specific or methylation-specific functions were examined, CLNMT displayed a pattern of inhibition which differs from that seen with calmodulin-activated enzymes. The product of calmodulin methylation, fully trimethylated calmodulin, and nonmethylatable VU-3 calmodulin acted as competitive inhibitors of CLNMT, with Ki values of 310 and 400 nM, respectively. Of the 13 compounds tested, which are inhibitors of calmodulin-dependent cyclic nucleotide phosphodiesterase, only the calmodulin-binding domain from Ca2+/calmodulin-dependent kinase II, melittin, and calmidazolium were effective inhibitors of CLNMT and each exhibited a complex pattern of inhibition with Kis values of 21, 50, and 65 nM, respectively. The only potent methylation-specific inhibitor was S-adenosyl-L-homocysteine, which also displayed a complex pattern of inhibition.

Effects of lead administration on developing rat kidney. I. Glutathione S-transferase isoenzymes.

The effects of acute and chronic exposure to lead on glutathione S-transferase (GST) isoforms were determined in developing kidney in the rat. The ontogeny of glutathione S-transferase isoforms was characterized as were the effects of depletion of dietary calcium on glutathione S-transferase isoform profiles in control and lead-treated rats. In the acute exposure experiments, rats of 14 and 50 days of age received three daily injections of lead acetate (114 mg/kg) and in the chronic exposure studies, rats received lead acetate at doses ranging from 50 to 500 ppm in their drinking water. Lead acetate administration in these chronic studies began 1 day after conception. Acute and chronic lead exposure had similar effects, causing increases in all but one glutathione S-transferase isoform (Yb3); these increases were markedly exacerbated by dietary calcium depletion. In all lead paradigms, GST subunits Yb1 and Yp showed the largest increases--greater than 25-fold in rats fed a low-calcium diet. GST subunit Yb3 showed small increases in the 14-day acute lead and the 4 week low-calcium animals and did not increase in other groups. Lead-related increases in GSTs were partially reversed by transferring animals previously receiving lead to lead-free water for a 4-week period. Kidneys of rats fed the low-calcium diet did not have detectable GST Yk, but in rats on this low-calcium diet that received 500 ppm lead; this GST isoform was found at levels comparable to those in control rats fed lab chow.

Effects of lead administration on developing rat kidney. II. Functional, morphologic, and immunohistochemical studies.

The effects of chronic lead administration on renal function and cytoarchitecture and on the immunohistochemical localization of glutathione S-transferase (GST) isoenzymes were determined. Pregnant rats were given 250 ppm lead acetate in drinking water from conception until weaning and mothers and pups received 500 ppm of lead acetate from weaning until termination at either 3 or 7 weeks of age. Light and electron microscopic studies after 3 weeks of lead administration showed tubular injury with frequent mitoses noted in proximal tubular cells and, after 7 weeks of treatment, interstitial fibrosis, characteristic intranuclear inclusions, and tubular injury characterized by both nuclear and cytoplasmic pleomorphism. Rats treated with lead for 7 weeks showed significantly lower body weights and creatinine clearances than age-matched control animals. Immunohistochemical studies of glutathione transferase subunits in control rats showed unique isoform localization in each segment of the nephron; treatment with lead caused large increases in immunoreactive protein of Yc, Yk, Yb1, and Yp GST subunits in proximal tubules. No increases in the antioxidant enzymes copper-zinc superoxide dismutase, catalase, and glutathione peroxidase were found in lead-treated rats, but there was a diffuse lead-related increase in immunoreactive protein for manganese superoxide dismutase throughout the renal cortex. Our results demonstrate large lead-induced increases of specific isoforms of glutathione S-transferase in specific kidney cell types and show that these increases preceded irreversible renal damage.

Concomitant preparative isolation of calmodulin and heat shock protein (hsp90) from bovine testes.

We have described a convenient procedure for isolating large amounts of both calmodulin and the 90-kDa heat shock protein (hsp90) from bovine testes. These two proteins coeluted from phenyl-Sepharose during calcium-dependent hydrophobic interaction chromatography. The hsp90 was separated from calmodulin by gel filtration, purified by reverse-phase HPLC or lectin affinity chromatography, and identified by N-terminal amino acid sequence and immunoblotting with hsp90-specific monoclonal antibody. The N-terminus of bovine hsp90 shared 93% amino acid identity with the murine alpha-isoform of hsp90. A third major testes protein of 97 kDa was also isolated with this protocol and was found to be homologous to the 94-kDa glucose-regulated protein.

Glutathione S-transferase isoenzymes in rat brain neurons and glia.

The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes and a structurally unrelated microsomal enzyme that is involved in the detoxication of electrophilic xenobiotics. These enzymes also participate in the intracellular binding and transport of a broad range of lipophilic compounds including bilirubin, and hormones such as the glucocorticoids and thyroid hormones. The present investigation demonstrates that GSTs are present in neurons of the brainstem, forebrain, and cerebellum. An isoenzyme-specific distribution of GSTs was found in cytoplasm, nuclei, and nucleoli. The regional and cellular distribution of cytosolic GSTs in the brain was studied by immunohistochemistry, spectrophotometric enzyme assay, and reverse-phase HPLC. Polyclonal antibody against microsomal GST was strongly reactive with Purkinje cells throughout the cerebellar cortex, and with neurons in the brainstem and hippocampus. Nuclei of Purkinje cells and of neurons in the brainstem, hippocampus, and cerebral cortex were immunopositive for alpha-class GST 1-1 (YaYa), whereas alpha-class GST 2-2 (YcYc) antibody was consistently immunoreactive with the nucleolus, but not with the nucleus or soma. All alpha-class GST antibodies studied were reactive, to various degrees, with astrocytes and choroid plexus; however, ependymal cells of the subventricular zones were immunonegative. alpha-class GST 8-8 (YkYk) immunoreactivity was specifically localized to endothelial cells and/or astrocytic end feet associated with blood vessels. Reverse-phase HPLC indicated that there were also substantial regional differences in the pattern of alpha-, mu-, and pi-class GST subunit expression. For example, the thalamus/hypothalamus had the highest GST activity and greatest concentration of total GST protein and mu-class GST subunit 6 (Yb3), whereas the brainstem had the greatest concentration of pi-class GST subunit (Yp). This regional variation in GST expression may be reflective of regional differences in cell populations. In cerebellar cortex, the concentration of mu-class GST subunit 4 (Yb2) was greatest in the flocculus and lowest in the vermis. This is of clinical interest because the pattern of expression of mu-class GST subunit 4 (Yb2) in the cerebellum coincides with the known regional susceptibility of this structure to degeneration after exposure to toxic or metabolic insults. The vermis is most susceptible to these insults, whereas the lateral lobes and flocculus are most resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein methylation in cerebellar synaptosomes.

Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21-26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21-26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21-26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21-26-kDa family was identified as GTP-binding proteins by [alpha-32P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [alpha-32P]GTP. When synaptosomes were screened for putative methyltransferases by S-adenosyl-L-[methyl-3H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.

Effects of hyperbilirubinaemia on glutathione S-transferase isoenzymes in cerebellar cortex of the Gunn rat.

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue distribution of enzymic methylation of glutathione S-transferase and its effects on catalytic activity. Methylation of glutathione S-transferase 11-11 inhibits conjugating activity towards 1-chloro-2,4-dinitrobenzene.

Glutathione S-transferases (GSTs) were isolated from rat liver, lung, heart, kidney, testis and brain by coupled affinity chromatography and subunits were resolved by reverse-phase h.p.l.c. The reverse-phase h.p.l.c. technique was improved from our previously published work [Johnson, Neal, Collins & Siegel (1990) Biochem. J. 270, 483-489] by changing from a C4 to a C18 wide-pore reverse-phase column; this resulted in baseline or near-baseline resolution of all GST subunits. There were significant tissue-dependent differences in the expression of GST subunits and the level of GST subunits present was quantitatively determined for each of the tissues. The extent of methylation of GSTs in vitro and distribution of GST methyltransferase (GST-MT) was determined in cytosolic fractions from each of these tissues. Purified GST isoenzymes were methylated with partially purified liver GST-MT. Methylation of Mu class subunits 3 and 4, the preferred substrates of methylation in liver, was substoichiometric in all tissues. The extent of methylation of subunit 3 ranged from 0.13% to 0.94% and subunit 4 from 0.03% to 0.60%. Methylation of Alpha class subunits was either not detectable or 5-10-fold less than that of Mu class subunits 3 and 4. Pi class subunit 7 was methylated to a greater extent than the Alpha class subunits but less than Mu class isoenzymes. A notable exception to this low level of methylation was GST 11-11, found mainly in testis and brain. Methylation of subunit 11 reached 21.9% (219 pmol of methyl group/nmol of subunit 11) when this isoenzyme was incubated with partially purified liver GST methyltransferase. Methylation of GST 11-11 was found to inhibit the conjugating activity of this isoenzyme towards 1-chloro-2,4-dinitrobenzene; the degree of inhibition of conjugating activity correlated with the extent of methylation of GST 11-11. GST-MT activity toward GST subunits 3, 4 and 11 was present in kidney and liver, detectable in lung and heart, but absent from brain and testis. Anion-exchange chromatography of GST-MTs from liver and kidney suggested the presence of four different forms of GST-MT (I-IV) and indicated that GST-MT isoenzymes III and IV were present at significantly lower concentrations in kidney than liver. The present paper shows that methylation is an enzyme-catalysed reaction that differs in substrate-specificity with respect to different GST isoenzymes, that expression of GST-MT is tissue-dependent and multiple forms of the enzyme are present in liver and kidney, and that methylation inhibits GST activity.(ABSTRACT TRUNCATED AT 400 WORDS)

A calmodulin endoproteinase from mitochondrial membranes.

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.