Tohru Nishinaka

Characterization of multiple forms of carbonyl reductase from chicken liver

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.

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.

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).

Unique Dihydrodiol Specific Dehydrogenase of Bovine Liver: Inhibition Studies and Comparison with Aldo/Keto Reductase

Dihydrodiol dehydrogenase (EC 1.3.1.20: DD) catalyzes the dehydrogenation of the dihydrodiols of benzo(a)pyrene and benzo(a) anthracene in the presence of NADP+ and forms ortho-qninone (Vogel et al., 1980; Smithgall et al., 1988a). We previously reported on the purification of three multiple forms of bovine liver DD, DD1, DD2 and DD3, using benzenedihydrodiol Urcms-1,2-dihydrobenzene-1,2-diol) which is a model substrate for DD with NADP+ as a coenzyme (Nishinaka et al., 1991). DDl and DD2 have been identified with 3α-hydroxysteroid dehydrogenase (Nanjo et al., 1992) and high-ifm aldehyde reductase (Terada et al., 1985), respectively. On the other hand, DD3 is a unique enzyme which can specifically catalyze the dehydrogenation of benzenedihydrodiol and naphthalenedihydrodiol and has no activity toward other alcohols, aldehydes, ketones and quinones which are well known substrates for aldo/keto reductases. The Km-value of DD3 for benzenedihydrodiol was the lowest among the three enzymes. Thus, DD3, judging from its substrate specificity and inhibitor sensitivity, could not be classified into any known DD including carbonyl and aldehyde reductases and steroid dehydrogenases (Balcsak et al., 1983; Hara et al., 1986; Sawada et al., 1989). The most interesting property of DD3 is an immunological crossreactivity with DDl. Properties of bovine liver cytosolic DD were summarized in Table 1.