Yorishige Imamura

Binding of pirprofen to human serum albumin studied by dialysis and spectroscopy techniques

The interaction of pirprofen with human serum albumin (HSA) was investigated by equilibrium dialysis and spectroscopic (UV absorption, fluorescence, CD, NMR) techniques. It was found that HSA binds pirprofen nonstereospecifically. The binding of pirprofen depends upon the N-B conformational change of albumin. Chloride ions appear to displace the drug from its binding site. The thermodynamic parameters suggest that the interaction may be explained by electrostatic as well as hydrophobic forces. The absorption spectral changes which accompanied the binding of pirprofen to HSA implied that the aromatic portion of drugs was inserted into the hydrophobic crevice in the protein, while the carboxyl group of the drug interacted with a cationic site on the albumin surface. The NMR data indicated that the pyrroline ring and propionic acid parts may be the major binding site for HSA. A specific binding site for pirprofen on the HSA was found to be site II, benzodiazepine site, using fluorescence probes and drug markers. In addition, from the binding data with modified HSA, it seems that Tyr-411 is specifically involved in pirprofen binding.

Characterization of human DHRS4: An inducible short-chain dehydrogenase/reductase enzyme with 3β-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

A comparative study of the interaction of warfarin with human α1‐acid glycoprotein and human albumin

The interaction of warfarin with human α1‐acid glycoprotein (α1‐AGP) and human albumin (HA) has been investigated using fluorescence and circular dichroism techniques. The fluorescence of warfarin is greatly enhanced following binding to α1‐AGP or HA, the binding constant for a single site being estimated by the Scatchard method. The binding constants for the two serum proteins are similar, but the thermodynamic parameters differ. The binding constants increase as the pH is raised to 9ṁ0. Various basic drugs, such as chlorpromazine, propranolol and imipramine, markedly inhibited the binding of warfarin to α1‐AGP. But, some acidic drugs, including phenylbutazone, effectively displaced warfarin bound to HA. The difference in CD spectra observed for α1‐AGP and HA indicated that the drug‐binding sites of the two proteins might have different asymmetries. It thus appears that the mode of interaction of warfarin with the two proteins differs.

Molecular Basis for Peroxisomal Localization of Tetrameric Carbonyl Reductase

Pig heart peroxisomal carbonyl reductase (PerCR) belongs to the short-chain dehydrogenase/reductase family, and its sequence comprises a C-terminal SRL tripeptide, which is a variant of the type 1 peroxisomal targeting signal (PTS1) Ser-Lys-Leu. PerCR is imported into peroxisomes of HeLa cells when the cells are transfected with vectors expressing the enzyme. However, PerCR does not show specific targeting when introduced into the cells with a protein transfection reagent. To understand the structural basis for peroxisomal localization of PerCR, we determined the crystal structure of PerCR. Our data revealed that the C-terminal PTS1 of each subunit of PerCR was involved in intersubunit interactions and was buried in the interior of the tetrameric molecule. These findings indicate that the PTS1 receptor Pex5p in the cytosol recognizes the monomeric form of PerCR whose C-terminal PTS1 is exposed, and that this PerCR is targeted into the peroxisome, thereby forming a tetramer.

Cloning, expression and tissue distribution of a tetrameric form of pig carbonyl reductase.

In this study, we isolated a cDNA for tetrameric carbonyl reductase (CR) from pig heart. The pig CR showed high amino acid sequence identity (81%) with rabbit NADP(+)-dependent retinol dehydrogenase (NDRD). The purified recombinant pig CR and NDRD were about 100-kDa homotetramers and exhibited high reductase activity towards alkyl phenyl ketones, alpha-dicarbonyl compounds and all-trans-retinal. The identity of NDRD with the tetrameric CR was verified by protein sequencing of CR purified from rabbit heart. Both tetrameric CR and its mRNA were ubiquitously expressed in pig and rabbit tissues. The pig and rabbit enzymes belonged to the short-chain dehydrogenase/reductase family, and their sequences comprise a C-terminal SRL tripeptide, which is a variant of the type 1 peroxisomal targeting signal, SKL. Transfection of HeLa cells with vectors expressing pig CR demonstrated that the enzyme is localized in the peroxisomes. Thus, the tetrameric form of CR represents the first mammalian peroxisomal enzyme that reduces all-trans-retinal as the endogenous substrate.

(R)-ACX is a novel sufonylurea compound with potent, quick and short-lasting hypoglycemic activity.

We investigated the mechanism of the hypoglycemic effect of (R)-4-(1-acetoxyethyl)-N-(cyclohexylcarbamoyl)benzene-sulfonamide [(R)-acetoxyhexamide; (R)-ACX], a new sulfonylurea compound. (R)-ACX potently stimulated the release of insulin from cultured pancreatic beta-cells (HIT T15 cells), established from hamster islet cells SV40-transformed. When (R)-ACX was orally administered to fasted rats, it decreased the plasma glucose level in a dose-dependent manner. The hypoglycemic effect of (R)-ACX was quick and short lasting, as compared to that of acetohexamide and glibenclamide. The quick and short-lasting hypoglycemic effect of (R)-ACX was thought likely to result from rapid absorption of (R)-ACX and rapid elimination of (R)-ACX and its metabolite, (R)-hydroxyhexamide. Furthermore, (R)-ACX was found to suppress the increase of blood glucose level due to starch loading in fasted mice. (R)-ACX may be useful in the control of postprandial hyperglycemia to patients with non-insulin-dependent diabetic mellitus.

Stereoselective reduction of 4-benzoylpyridine by recombinant pig heart carbonyl reductase

Abstract Optically active (−)- and (+)-α-phenyl-4-pyridylmethanol [(−)- and (+)-PPOL] were synthesized from 4-benzoylpyridine (4-BP) and established to possess (S)- and (R)-configurations, respectively, by X-ray single crystal structure analysis. When 4-BP was incubated in the reaction mixture containing recombinant pig heart carbonyl reductase (rPHCR) and NADPH, the reduction product appeared as a main peak corresponding to (S)-(−)-PPOL on high-performance liquid chromatography (HPLC). This result indicates that rPHCR has the ability to reduce stereoselectively 4-BP to (S)-(−)-PPOL with high optical purity.

Kinetic studies on the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney

The kinetic mechanism for the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney was investigated. The initial velocity and product inhibition studies indicated that the enzymatic reaction follows an ordered Bi Bi mechanism, in which NADPH binds to the enzyme first and NADP leaves last. This kinetic mechanism was confirmed on the basis of the dead-end inhibition by Cibacron Blue and the binding of NADPH and NADP to the free enzyme. However, whether or not coenzyme-induced isomerization is involved in the enzymatic reaction remains to be clarified. In kinetic studies of inhibition of the enzyme by therapeutically active drugs, indomethacin and befunolol were found to be noncompetitive and competitive inhibitors, respectively, with respect to acetohexamide.

A possible mechanism of resistance to cadmium toxicity in male Long-Evans rats

The susceptibility to cadmium (Cd)-induced toxicity in male Long-Evans (LE) rats was compared with that in male Fischer 344 (Fischer) and Wistar-Imamichi (WI) rats, which are sensitive and resistant, respectively, to Cd toxicity. All rats of the LE and WI strains survived for 7 days after the treatment with a toxic dose of Cd (6.5mg/kg b.w.). However, all rats of the Fischer strain died by the following day. The strong resistance to Cd toxicity in the LE strain was confirmed to be independent of metallothionein synthesis induced by Cd. The hepatic and renal Cd contents after its administration were significantly lower in the LE strain than in the Fischer strain. Furthermore, the hepatic and renal zinc (Zn) contents after its administration were significantly lower in the LE strain than in the Fischer strain. These limited data suggest that the strong resistance to Cd toxicity in male LE rats results from, at least in part, the lower accumulation of the metal in the liver and kidney, in a similar mechanism as the lower Zn accumulation.

Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver.

The further metabolism of N-desisopropylpropranolol (NDP), a side-chain metabolite of propranolol (PL), was investigated in isolated rat hepatocytes. Propranolol glycol (PGL) was generated from NDP as a major metabolite. Naphtetrazole (NTE), a potent inhibitor of monoamine oxidase (MAO), significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde (4-NBA), a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP [S(-)-form of NDP] was larger than that of PGL formed from R-NDP [R(+)-form of NDP].