| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
(1) Studies on human vitamin D_3 lα-hydroxylase (CYP27B1)
Human CYP27B1 cDNAs derived from normal subject, patients of pseudovitamin D deficiency rickets (PDDR) and mutants obtained with site-directed mutagenesis were each expressed in E.coli. Their enzymatic properties together with spectrometric properties of the corresponding mutants of CYP27A1 revealed the functions of the mutated amino acid residues of CYP27B1. Co-expression of CYP27B1 with adrenodoxin and NADPH-adrenodoxin reductase in E.coli remarkably enhanced 1a-hydroxylase activity toward 25-hydroxyvitamin D_3 (25(OH)D_3). The recombinant E.coli could be practically useful as a bio-catalyst to produce 1α, 25- dihydroxyvitamin D_3 (1α, 25(OH)_2D_3).
(2) Studies on human vitamin D_3 24-hydroxylase (CYP24)
Human CYP24 produced in E.coli showed remarkable metabolism towards 25(OH)D_3 and 1α, 25(OH)_2D_3. When the substrate 25(OH)D_3 was added to the membrane fraction prepared from the recombinant E.coli cells, four metabolites in C-
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24 hydroxylation pathway and three metabolites in C-23 hydroxylation pathway were observed. On the other hand, rat CYP24 showed only 24-metabolic pathway. These results suggest that CYP24 has the species difference in the enzymatic properties, and the E.coli expression system for human CYP24 is useful for predicting the metabolism of vitamin D analogs before clinical trials.
(3) Studies on human vitamin D_3 25-hydroxylase (CYP27A1)
Human CYP27A1 cDNA was expressed in E.coli and its enzymatic properties were revealed. Surprisingly, eight forms of metabolites of vitamin D_3 including the major product 25(OH)D_3 were detected in the recombinant system containing CYP27A1. These results suggest that human CYP27A1 catalyzes multiple reactions and multi-step metabolism towards vitamin D_3. Kinetic studies have evaluated a physiological meaning of each reaction catalyzed by CYP27A1.
(4) Metabolism of vitamin D_3. and its analogs in human body
The studies on human CYP27A1, CYP27B1 and CYP24 could explain more than thirty metabolites of vitamin D_3. detected in human body. The E.coli expression systems for human CYP27A1, CYP27B1 and CYP24 established in this study appear useful as bio-catalysts to produce active forms of vitamin D_3 such as 1α, 25(OH)_2D_3, and also useful to predict the metabolism of vitamin D analogs before clinical trials. Less
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