Hirofumi Nanjo

Pig lens glutathione S-transferase belongs to class Pi enzyme

Class Pi glutathione S-transferase was purified to homogeneity from pig lens cytosol. This enzyme was composed of two identical 22 kDa subunits and had isoelectric point of 8.5 from the results of SDS gel electrophoresis, gel filtration, amino acid sequence analysis and isoelectric focusing. Amino acid sequence of N-terminal 15 residues was almost identical to class Pi enzymes from human, rat and mouse. Antibody against the pig enzyme crossreacted to human glutathione S-transferase-pi and anti-rat glutathione S-transferase-P antibody crossreacted to pig enzyme.

Characterization of bovine liver cytosolic 3α-hydroxysteroid dehydrogenase and its aldo-keto reductase activity

1. 3 alpha-Hydroxysteroid dehydrogenase was purified to homogeneity from bovine cytosolic fraction, which was monomeric and its molecular weight was estimated to be about 35 kDa. 2. The enzyme had ability to catalyze NADP(H)-dependent oxidoreduction of position 3 alpha-hydroxy and keto group of steroids and also could catalyze the reduction of some ketones and quinones. 3. In addition, benzenedihydrodiol was one of the substrates of dehydrogenase activity with NADP+. 4. Indomethacin, synthetic steroids and SH-reagents were potent inhibitors for this enzyme. 5. Inactivation of the enzyme by GSSG-treatment was restored to its original activity by the addition of DTT. 6. The presence of coenzyme, 0.33 mM NADP+, completely protected from the DTNB-inactivation. 7. Bovine liver cytosolic enzyme immunologically crossreacted with rat liver 3 alpha-hydroxysteroid dehydrogenase.

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase - reaction mechanism and bile acid dehydrogenase activity

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Cloning and Expression of cDNA Encoding Bovine Liver Dihydrodiol Dehydrogenase 3, DD3

Dihydrodiol dehydrogenase (DDH) [EC 1.3.1.20] has been suggested to play an important physiological role in the metabolic detoxication of polycyclic aromatic hydrocarbons (PAHs) (Glatt et al., 1979). The pathways for metabolic activation and detoxication of a typical PAH, benzo[a]pyrene, are shown in Scheme 1. Environmental PAH is mainly activated through the action of liver P-450. The resulting 7,8-epoxide moiety is hydrolyzed by epoxide hydrolase in microsome. Then, the 7,8-dihydrodiol of PAH is further oxygenated to the 7,8-dihydrodiol-9,10-epoxide form by P-450 (Penning, 1993). This compound is assumed to be an ultimate carcinogen which binds covalently to DNA. Alternatively, DDH is reported as an important converting enzyme of the 7,8-dihydrodiol moiety to less active 7,8-dione (dicarbonyl). The dicarbonyl compound is further detoxicated with nonenzymatic ghlutathione-conjugation reaction (Penning, 1993).

Comparison of purified lens glutathione S-transferase isozymes from rabbit with other species.

Two glutathione S-transferase (GST) isozymes, GST-rl1 and GST-rl2, were purified from rabbit lenses and their properties were compared with those of other animals. GST-rl1 and GST-rl2 are dimeric enzymes whose subunit sizes are 24,000 and 21,500, respectively. The substrate specificities and inhibitor sensitivities of GST-rl1 and GST-rl2 are different from each other and from those of the isozymes from other animals. GST-rl1 immunologically crossreacted with the antibody against class mu GST (rat GST Yb1-Yb1), and GST-rl2 crossreacted with the antibody against class pi GST (rat GST Yp-Yp). N-Terminal amino acid sequences of GST-rl1 and GST-rl2 have great homology with other class mu and class pi enzymes, and thus indicate that they belong to class mu and class pi, respectively. Class pi GST-rl2 is inactivated by 1,2-naphthoquinone, an oxidized metabolite of naphthalene, but class mu GST-rl1 is insensitive to it. These results are similar to those of class pi pig lens GST and class mu bovine lens GST. Thus, the expression pattern of GST isozymes in lens varies with animal species, and may relate to their variation in sensitivity to oxidative stress.

Study on Dihydrodiol Dehydrogenase (I) Molecular Forms of the Enzyme and the Presence of a Dihydrodiol Specific Enzyme in Bovine Liver Cytosol

It has been known that benzo(a)pyrene and benzo(a)anthracene, typical carcinogenic polycyclic aromatic hydrocarbons, are metabolized in microsome to the corresponding dihydrodiols via epoxides (Yang, et al., 1976; Thakker, et al., 1982) and then converted to the ultimate carcinogens (Buening, et al., 1978). Dihydrodiol dehydrogenase is an enzyme which catalyzes the dehydrogenation of the dihydrodiols of benzo(a)pyrene and benzo(a)anthracene in the presence of NADP+ and forms o-quinone (Vogel, et al., 1980; Smithgall, et al., 1988). The addition of this enzyme to the Ames test significantly reduced the mutagenicity of benzo(a)pyrene, suggesting that this enzyme might detoxify the trans-dihydrodiols which were formed in situ by oxidizing them to the less reactive o-quinones (Glatt, et al., 1979). In addition, similar experiments showed that the purified enzyme reduced the mutagenicity of other polycyclic aromatic hydrocarbons (Smithgall, et al., 1986). On the basis of these facts, Penning and coworkers suggested that dihydrodiol/ 3α -hydroxysteroid dehydrogenase might play an important role in the detoxification of these carcinogenic polycyclic aromatic hydrocarbons in rat liver (Smithgall, et al., 1988). Recently, many dihydrodiol dehydrogenases were purified from various animals and tissues, and these enzymes were identified as 3α -hydroxysteroid dehydrogenase, 17β -hydroxysteroid dehydrogenase and aldehyde reductase from their substrate specificities and inhibitor sensitivities (Smithgall, et al., 1988; Sawada, et al., 1988; Terada, et al., 1990).

Study on Dihydrodiol Dehydrogenase (II) Modulation of Dihydrodiol Dehydrogenase Activity by Biological Disulfides

Rat liver cytosolic 3α-hydroxysteroid dehydrogenase (EC 1.1.1.50) (dihydrodiol dehydrogenase) which can catalyze the conversion between androsterone and androstanedione in the presence of NADP(H) has also been shown to catalyze the oxidation of benzenedihydrodiol to catechol (Penning et al, 1984, 1985; Hara et al., 1988). From the facts that can oxidize the trans-dihydrodiol of polycyclic aromatic hydrocarbons such as benzo(a)pyrene and benzo(a)anthracene, it has been suggested that dihydrodiol dehydrogenase plays an important role in the detoxification of the polycyclic aromatic hydrocarbon through the effective suppression in the formation of the ultimate carcinogenic anti-diol epoxides (Penning et al., 1984, 1985).

Mutational analyses of cysteine residues of bovine dihydrodiol dehydrogenase 3.

The cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase 3 (DD3) cDNA (1330 bp in full length) using the pKK223-3 expression vector has been reported previously. Recombinant DD3 (rDD3) was characterized in terms of its substrate specificity and inhibitor sensitivity [Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-553]. The nucleotide sequence of DD3 cDNA completely matched with that of bovine liver-type prostaglandin F synthase [Suzuki et al., J. Biol. Chem. 274 (1999) 241-248]. In the present study, we succeeded in high level expression of rDD3 in Escherichia coli BL21 (DE3) using the pET28a expression vector. rDD3 was easily and quickly purified to apparent homogeneity by one-step column chromatography using Ni(2+)-affinity resin. Furthermore, rDD3 showed essentially the same substrate specificity and inhibitor sensitivity to that of purified liver DD3. To analyze the role of cysteines (145, 154, 188, 193 and 206) in the enzymatic activity of DD3, site-directed mutagenesis of DD3 using the polymerase chain reaction method was performed. Mutants (C145S, C154S, C188S, C193S and C206S) were analyzed for substrate specificity, cofactor binding and inactivation by disulfide (dithio-bis(2-nitrobenzoic acid), alkylating reagent (N-ethylmaleimide) and oxidants (naphthoquinone and H(2)O(2)) Results indicated that these five cysteines of rDD3 may not be directly involved in substrate or cofactor binding. Mutant C193S showed strong resistance to SH-reagents unlike wild-type DD3 (WT) or other mutants. Both the WT and the other mutants showed essentially the same sensitivity to SH-reagents. Cofactor (NADP(+)) protected mutants C145S, C188S and C206S from inactivation as well as WT, while NAD(+) was not protective. Our present results indicate that Cys193, which is located close to the NADP(+)-binding site, may be involved in the alteration of enzymatic activity.

Characterization of two μ class glutathione S-transferases from guinea pig lens

Glutathione S-transferase (GST) plays an important role in the detoxifications of foreign electrophiles. Two GSTs of class mu from guinea pig lens were purified with Sephacryl S-100 gelfiltration, S-Hexyl glutathione Agarose affinity and Q-Sepharose anion exchange chromatographies. These GSTs (GST-A and B) showed similar relative molecular masses of 22.9 and 22.5 kDa, respectively. Two protein bands which crossreacted with anti GSTYb1 (GST 3-3) were detected in lens cytosolic crude extract on Western blotting and they showed Mrs corresponding to the purified enzymes. These GSTs showed a strong resistance against H2O2, 1,2-naphthoquinone and superoxide anion consistent with the other GSTs in class mu from animal tissues.

Irreversible inactivation of glutathione S-transferase-π by a low concentration of naphthoquinones.

π-Class glutathione S-transferase (GST-π) was very potently inactivated by oxidants such as H2O2, xanthine-xanthine oxidase and naphthoquinones. Thiols and glutathione analogs including dithiothreitol, reduced gluta-thione, cysteine, cysteamine, S-methyl-SG, S-hexyl-SG and S-decyl-SG protected GST-π from the inactivation, but a substrate analog (2,4-dinitrophenol), superoxide dismutase and catalase did not, suggesting that the cysteinyl residue(s) in/nearby the glutathione binding site (G-site) may be oxidatively modified by these oxidants. Many reductants and radical scavengers including butylated hydoxytoluene, α-tocopherol, ascorbate, uric acid, mannitol, tyrosine, tryptophan, histidine, quercitrin and bilirubin had no effect on the inactivation. GST-π pretreated with cystamine was reactivated very efficiently by 50 mM DTT following incubation with 1,2-naphthoquinone, whereas cystamine-untreated GST-π was not reactivated.