Biomedical subjects

Shinichi Ikushiro

Publications and source records attributed to Shinichi Ikushiro.

4 recordsLinked to original sources

PubMed2006

Enzymatic properties of human CYP2W1 expressed in Escherichia coli.

The human genome project revealed a new member of the P450 family 2, CYP2W1, which has orthologous form in other vertebrate species, suggesting CYP2W1's significant physiological function. Recently, it was reported that CYP2W1 can metabolize arachidonic acid. In this study, we isolated human CYP2W1 cDNA, and successfully expressed truncated CYP2W1 lacking N-terminal 20 amino acids in Escherichia coli cells. In the bicistronic expression system for human CYP2W1 and NADPH-P450 reductase, the formation of blue pigment, indigo, was observed in bacterial cultures. Based on this result, we revealed that CYP2W1 catalyzes the oxidation of indole. In addition, CYP2W1 showed monooxygenase activity towards 3-methylindole and chlorzoxazone. However, no activity was observed towards fatty acids including arachidonic acid. Further analysis using an E. coli expression system will reveal substrate specificity of CYP2W1 and why this P450 isoform is universally conserved in vertebrates.

Amino Acid Sequence↗

PubMed2006

Interaction between mitochondrial CYP27B1 and adrenodoxin: role of arginine 458 of mouse CYP27B1.

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2005

Decrease in serum thyroxine level by phenobarbital in rats is not necessarily dependent on increase in hepatic UDP-glucuronosyltransferase.

We have previously reported that there is a poor correlation between increase in the levels of UDP-glucuronosyltransferases, UGT1A1 and UGT1A6, and decrease in the levels of serum total thyroxine (T4) and free T4 in phenobarbital (PB)-treated rats, although the PB-induced decrease in rats is generally thought to occur through induction of the UDP-glucuronosyltransferase (T4-UDP-GT: UGT1A1 and UGT1A6). In the present study, to clarify a relationship between the decrease in serum T4 level and the increase in the T4-UDP-GT activity by PB in rats, we examined the relationship using Gunn rats, a mutant strain of Wistar rats deficient in UGT1A isoforms. Levels of serum total T4, free T4, and total triiodothyronine (T3) were markedly decreased not only in Wistar rats but also in Gunn rats 1 day after the final administration of PB (80 mg/kg i.p., once daily for 4 days), and no significant difference in magnitude of the decrease between Wistar and Gunn rats was observed. On the other hand, the level and activity of T4-UDP-GT were significantly increased by treatment with PB in Wistar rats but not in Gunn rats. Furthermore, significant decrease in the activity of hepatic type I iodothyronine deiodinase, which mediates the deiodination of T4 and T3, by PB treatment was observed in both Wistar and Gunn rats. In addition, no significant change in the level of serum thyroid-stimulating hormone, the activity of hepatic sulfotransferase, and the binding of [125I]T4 to serum transthyretin and albumin by PB treatment was observed in either Wistar or Gunn rats. In conclusion, the present results demonstrate that the decrease in serum total T4 level by PB in Gunn rats is not dependent on the increase in hepatic T4-UDP-GT activity and suggest that even in Wistar rats, the PB-induced decrease in serum T4 level does not occur only through increase in hepatic T4-UDP-GT.

Animals↗

PubMed2004

A possible mechanism for decrease in serum thyroxine level by polychlorinated biphenyls in Wistar and Gunn rats.

We have previously demonstrated that in mice, the decrease in serum thyroxine (T(4)) level by polychlorinated biphenyls (PCBs) occurs without an increase in the UDP-glucuronosyltransferase (T(4)-UDP-GT) for T(4) glucuronidation, although the PCB-induced decrease in rats is generally thought to occur through induction of T(4)-UDP-GT, UGT1A1, and UGT1A6. In the present study, to further clarify the relationship between the decrease in serum T(4) level and the increase in UGT1A activity by PCB in rats, we examined the relationship using Wistar rats and Gunn rats, a mutant strain of Wistar rats deficient in UGT1A isoforms. The serum total T(4) level was markedly decreased not only in the Wistar rats but also in the Gunn rats 4 days after treatment with a PCB, Kanechlor-500 (KC500, 100 mg/kg) or 2,2',4,5,5'-pentachlorobiphenyl (PentaCB, 112 mg/kg), and there was no significant difference in magnitude of the decrease between the two rat strains. At the same time, the level and activity of T(4)-UDP-GT were significantly increased by treatment with either KC500 or PentaCB in Wistar rats but not in Gunn rats. In addition, no significant change in the level of serum total triiodothyronine (T(3)) and thyroid-stimulating hormone by the KC500 treatment was observed in either Wistar or Gunn rats. Furthermore, significant decrease in the activity of hepatic type-I deiodinase, which mediates the deiodization of T(4) and T(3), by treatment with KC500 or PentaCB was observed in both Wistar and Gunn rats. From the serum of KC500- or PentaCB-treated Wistar and Gunn rats, mono- and di-hydroxylated PCB metabolites, which would bind to T(4) binding serum protein (transthyretin), were detected. In conclusion, the present results suggest that the decrease in serum total T(4) level by either KC500 or PentaCB in Gunn rats was not dependent on the increase in hepatic T(4)-UDP-GT activity. The findings further suggest that the PCB-mediated decrease in serum T(4) level might occur, at least in part, through formation of the hydroxylated PCB metabolites. Furthermore, even in Wistar rats, the PCB-mediated decrease in serum T(4) level might occur not only through the increase in hepatic T(4)-UDP-GT but also via formation of hydroxylated PCB metabolites.

Animals↗