Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism.

To provide a molecular basis for understanding the possible mechanism of action of antidipsotropic agents in laboratory animals, aldehyde dehydrogenase (ALDH) isozymes were purified and characterized from the livers of hamsters and rats and compared with those from humans. The mitochondrial ALDHs from these species exhibit virtually identical kinetic properties in the oxidation and hydrolysis reactions. However, the cytosolic ALDH of human origin differs significantly from those of the rodents. Thus, for human ALDH-1, the Km value for acetaldehyde is 180 +/- 10 micromolar, whereas those for hamster ALDH-1 and rat ALDH-1 are 12 +/- 3 and 15 +/- 3 micromolar, respectively. Km values determined at pH 9.5 are virtually identical to those measured at pH 7.5. In vitro human ALDH-1 is 10 times less sensitive to disulfiram inhibition than are the hamster and rat cytosolic ALDHs. Competition between acetaldehyde and aromatic aldehydes or naphthaldehydes for the binding and catalytic sites of ALDHs shows their topography to be complex with more than one binding site. This also follows from data on substrate inhibition and activation, effects of NAD+ on ALDH-catalyzed hydrolysis of p-nitrophenyl esters, substrate specificity toward aldehydes and p-nitrophenyl esters, and inhibition by disulfiram in relation to oxidation and hydrolysis catalyzed by the ALDHs. The data further suggest that acetaldehyde cannot be considered as a "standard" ALDH substrate for studies aimed at aromatic ALDH substrates, e.g. biogenic aldehydes. Apparently, in human liver, only mitochondrial ALDH oxidizes acetaldehyde at physiological concentrations, whereas in hamster or rat liver, both the mitochondrial and cytosolic isozymes will do so.

Glutathione transferases of classes alpha, mu and pi show selective expression in different regions of rat kidney.

1. Glutathione transferases (GST) are mainly cytosolic and occur in multiple forms, which can be arranged in three distinct, structural classes. The different enzyme forms show distinct substrate specificities with electrophilic and genotoxic substances. The expression of the alpha subunits 1, 2 and 8, the mu subunits 3, 4 and 6, and the pi subunit 7 of GST in different parts of the rat kidney was determined immunohistochemically. 2. GST immunoreactivity was present predominantly in the nephron, collecting duct and urothelium. 3. A conspicuous finding was that subunits 1, 2 and 8 were localized to the proximal tubules, while the mu subunit 3 was demonstrable in epithelial tubular cells from the distal tubules to the urothelium. The immunoreactivity of subunits 4 and 6 could be visualized in epithelial cells from the ascending thin limb to the collecting ducts. Subunit 7 was found in the thin limb of the loop of Henle, and in scattered cells in the distal tubules. 4. The urothelial cells covering the papilla and the renal calyces showed immunoreactivity to GST subunits 2-4 and 6-8. 5. Thus, in the nephron the class alpha GSTs were selectively expressed in the proximal tubules and the class mu and class pi GST in the thin loop of Henle and distal tubules. The cells in the collecting ducts and the urothelium, which have a different ontogeny than the nephron, do not show any corresponding differential distribution of the GST classes. 6. Cells in a given location were in some cases found to be non-reactive with a given antiserum in an otherwise immunoreactive cell population, demonstrating a spatial variation in GST expression. The immunoreactivity to the different forms of GST was predominantly cytoplasmic but a nuclear localization could also be demonstrated. 7. The panel of antibodies to GST may tentatively be used as markers in localizing lesions in restricted parts of the nephrons and to elucidate dynamic alterations in the tubular system in response to physiological and toxic agents.

Denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is known to be detoxified by a denitrosation reaction catalyzed by glutathione-dependent enzymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., 11:175-176, 1983). Using a modification of their procedure, we have measured the ability of different purified rat glutathione transferase isoenzymes to denitrosate BCNU. The catalytic efficiencies of the isoenzymes for the denitrosation reaction expressed as the ratio of Vmax to Km were as follows (isoenzyme, Vmax/Km): 1-2, 2.3; 3-3, 12.2; 3-4, 29.2; and 4-4, 26.1. Thus, the class mu isoenzymes containing subunit 4 are by far the best catalysts of the BCNU denitrosation reaction. The class pi transferase 7-7 and class alpha transferases 1-1 and 1-2 demonstrated very weak catalytic activity with BCNU. Determination of the glutathione transferase isoenzyme profiles of 9L rat brain tumor cells and the BCNU-resistant 9L-2 subline by immunoblotting revealed that although the resistant 9L-2 cells contain lower total glutathione transferase activity than 9L cells, they have elevated levels of the class mu transferases. Also, the class pi transferases were found to be down-regulated in 9L-2 as compared with 9L cells. Thus, the increased resistance of 9L-2 cells to BCNU may, in part, be explained by up-regulation of class mu transferase expression with consequent increased capacity for BCNU detoxication. Further support for this hypothesis comes from the fact that pretreatment of 9L-2 cells with the glutathione transferase inhibitors ethacrynic acid or triphenyltin chloride enhanced the cytotoxic effects of BCNU. These results suggest that the class mu transferases play a role in the resistance of brain tumor cells to BCNU.

Glutathione transferase catalyzed conjugation of benzo[a]pyrene 7,8-diol 9,10-epoxide with glutathione in human skin.

Glutathione transferase (GST) activity towards racemic as well as the resolved enantiomers of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (anti-BPDE) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in post-microsomal supernatants (PMS) obtained from eight human skin samples. All preparations showed significant activity towards anti-BPDE and an almost exclusive preference for the more tumourigenic (+)-enantiomer. The specific activity towards (+)-anti-BPDE varied about five-fold between different PMS (range 147-781 pmol/min per mg protein) whereas the variation in specific activities towards CDNB was about two-fold (range 30-71 nmol/min per mg protein). The activities obtained with PMS at saturating concentrations of racemic anti-BPDE were about half of the activity towards the (+)-enantiomer indicating that (-)-anti-BPDE competitively inhibits conjugation of the (+)-form. No correlation was evident between the activities towards (+)-anti-BPDE and CDNB implying that different classes of GST isoenzymes participated in the two different reactions. Immunoblot analysis revealed the presence of Class Alpha and Pi isoenzymes whereas Class Mu isoenzymes seemed to be absent in the human skin samples analyzed. Quantitatively, the Class Pi isoenzyme(s) predominated in all skin samples and the amount of enzyme was about 1-3 micrograms GST Pi/mg PMS protein. The almost exclusive conjugation of (+)-anti-BPDE by PMS and previous results with GST Pi enzymes from human placenta suggested that this type of enzymes catalysed the conjugation reaction. The five-fold variation in specific activity towards (+)-anti-BPDE observed among the different PMS may be explained by individual differences in GST Pi content or by the presence of endogenous modifiers of GST activity towards the diol-epoxide.

Glutathione transferases in rat hepatoma cells. Effects of ascites cells on the isoenzyme pattern in liver and induction of glutathione transferases in the tumour cells.

Rat hepatoma cells grown intraperitoneally as an ascites tumour were analysed with respect to their contents of cytosolic glutathione transferases. In contrast with normal liver tissue, the hepatoma cells were dominated by the class Pi glutathione transferase 7-7. All the major hepatic enzyme forms were down-regulated to almost undetectable concentrations. Livers of rats bearing ascites-hepatoma cells expressed low, but significant, amounts of protein which, by electrophoretic and immunochemical properties, appeared identical with transferase 7-7. This enzyme is not detectable in normal hepatocytes. Treatment of rats with trans-stilbene oxide induced the expression of transferase 7-7 in the livers of normal rats as well as in hepatoma-cell-bearing animals. In addition, a 2-fold induction of transferase 7-7 was measured in the hepatoma ascites cells. No significant elevation of any other enzyme forms in the hepatoma cells was noted.

Isoenzymes of glutathione transferase in rat small intestine.

The major glutathione transferases in the rat small-intestine cytosol were isolated and characterized. The enzymes active with 1-chloro-2,4-dinitrobenzene as second substrate were almost quantitatively recovered after affinity chromatography on immobilized S-hexylglutathione. The different basic forms of glutathione transferase, which account for 90% of the activity, were resolved by chromatofocusing. Fractions containing enzymes with lower isoelectric points were not further resolved. The isolated fractions were characterized by their elution position in chromatofocusing, apparent subunit Mr, reactions with specific antibodies, substrate specificities and inhibition characteristics. The major basic forms identified were glutathione transferases 1-1, 4-4 and 7-7. In addition, evidence for the presence of a variant form of subunit 1, as well as trace amounts of subunits 2 and 3, was obtained. A significant amount of transferase 8-8 in the fraction of acidic enzyme forms was demonstrated by immunoblot and Ouchterlony double-diffusion analysis. In the comparison of the occurrence of the different forms of glutathione transferase in liver, lung, kidney and small intestine, it was found that the small intestine is the richest source of glutathione transferase 7-7.

Expression of class Pi glutathione transferase in human malignant melanoma cells.

The occurrence of glutathione transferase in human malignant melanoma cell lines and solid tumor material has been analyzed and compared with the enzyme composition in fibroblasts and naevus samples. All cells and tissues investigated contained essentially only the acidic class Pi glutathione transferase as demonstrated by SDS-PAGE and immunoblotting. The enzyme was purified from tumor material and characterized. Its intracellular concentration was significantly higher in all the melanoma cell preparations analyzed than in the non-malignant cells, supporting the view that the class Pi glutathione transferase may contribute to the drug resistance that is characteristic of malignant melanoma.

Purification and characterization of three distinct glutathione transferases from mouse liver.

Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.

Simple inhibition studies for distinction between homodimeric and heterodimeric isoenzymes of glutathione transferase.

Simple inhibition studies in which fractional velocity is measured as a function of inhibitor concentration were used to distinguish heterodimeric from homodimeric isoenzymes of glutathione transferase. Homodimeric isoenzymes from rat, mouse, and human tissues were shown to give graphs of fractional velocity versus the logarithm of inhibitor concentration that were characterized by a sigmoid curve shape and a maximal slope of -0.58 at 50% inhibition, characteristic for linear inhibition of monomeric or non-cooperative oligomeric enzymes. In contrast, inhibition curves for heterodimeric isoenzymes from rat liver displayed significant deviations from these characteristics. The basis for the identification of heterodimers was the finding that the kinetic properties of subunits were additive such that the inhibition curve of a heterodimeric isoenzyme could be predicted from those of the corresponding homodimers. The method should be valuable in the differentiation between the multiple forms of glutathione transferase in tissues not previously characterized. It is suggested that the method should be applicable for discrimination also in other isoenzyme families consisting of oligomeric structures of identical and nonidentical subunits.

Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties.

The major isoenzymes of cytosolic glutathione transferase (EC 2.5.1.18) from rat, mouse, and man are shown to share structural and catalytic properties that can be used for species-independent classification. Rat, mouse, and human isoenzymes were grouped with respect to amino-terminal amino acid sequences, after correlation of seven structures analyzed in the present investigation with structures determined earlier. The isoenzymes were also characterized by substrate specificities and sensitivities to inhibitors, and the data were subjected to pattern recognition analysis. In addition, the various isoenzymes were tested for cross-reactivity by immunoprecipitation with antibodies raised against rat and human transferases. The different types of data were clearly correlated and afforded an unambiguous division of the isoenzymes into three classes named alpha, mu, and pi. Each of the three mammalian species studied contains at least one isoenzyme of each class. It is suggested that the similarities of the isoenzymes in a class reflect evolutionary relationships and that the classification applies generally.

Isoenzymes of glutathione transferase in rat kidney cytosol.

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a 'new' form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing.

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.

Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver.

Cytosolic GSH transferases have been purified from rat lung by affinity chromatography followed by chromatofocusing. On the criteria of order of elution, substrate specificity, apparent subunit Mr, sensitivity to inhibitors, and reaction with antibodies, transferase subunits equivalent to subunits 2, 3, and 4, in the binary combinations occurring in liver, were identified. However, subunit 1 (and therefore transferases 1-1 and 1-2) was not detected. The most conspicuous difference is the presence in lung of a new form, eluting at pH 8.7, which is not detected in rat liver. This isoenzyme (transferase "pH 8.7") is characterized by its low apparent subunit Mr and high efficiency in the conjugation of glutathione with anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, considered the ultimate carcinogen of benzo(a)-pyrene.

Inhibitors for distinction of three types of human glutathione transferase.

A set of inhibitors that are useful for distinction of three types of human cytosolic glutathione transferase is presented. The near-neutral transferase is inhibited most effectively by Cibacron blue (I50 = 0.05 microM), the acidic transferase by Cibacron blue (I50 = 0.5 microM), and the basic transferase by tributyltin acetate (I50 = 0.1 microM). The use of any of these two compounds makes possible differentiation between all three types of human transferase.

Amylase isozymes in the mosquito, Culex tritaeniorhynchus.

A survey of laboratory stocks of Culex tritaeniorhynchus Giles has uncovered the presence of four electrophoretic variants and one apparent null allele of amylase. Linkage experiments with heterozygous males indicate that amylase is sex-linked. The gene sequence is w (white eye) - Amy (amylase) - M (sex), and the recombination frequencies are: w - Amy = 6.02%, Amy - M = 1.35% and w - M = 7.37%.