| Research Abstract |
Cytochrome P-450 HP (CYP4B1) cDNA was isolated from human placental cDNA library and the amino acid sequence was deduced from the nucleic acid sequence by Yokotani et al. The function of cytocrome P-450 HP as an enzyme is not known, because the protein, cytochrome P-450 HP,have not been investigated. In this study, the primary structure was analyzed, and expression of P-450 HP cDNA have been tried with yeast and E.coli expression systems to characterize the nature of cytochrome P-450 HP.
1. Amino acid sequences of cytochrome P-450 HP,P-450ka-1 (CYP4A6) and P-450cam (CYP101) were aligned, and six substrate recognition sites (SRS) of P-450 HP were estimated from those of P-450 cam. Estimated SRS 1 of cytochrome P-450 HP was located in the region of (A.A.110-121). We had showed that the region (A.A.58-123) of P-450ka-1 plays an important role for substrate specificity (unpublished data). The amino acid sequence of this region of cytochrome P-450HP is very variable compared with that of cyt
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ochrome P-450ka-1 (homology is 32%). This result indicates that cytochrome P-450 HP has different functions from omega-hydroxylation.
2. Expression of cytochrome P-450 HP cDNA with E.coli expression system was tried. 6bp, 72bp and 102bp from 5'terminal of cDNA which coded hydrophobic amino acids were trimmed to facilitate expression in E.coli. Cytochrome P-450 was not detected in membrane and soluble fractions of E.coli transformed with cytochrome P-450 HP expression vecters.
3. Expression of cytochrome P-450 HP cDNA with yeast expression system was performed. The reader sequense of cDNA was replaced with AAAAAA.Microsome fraction of transformant was prepared, and P-450 content was determined (less than 0.003 nmol/mg). Expressed P-450 HP showed no catalytic activities toward various substrates.
4.8 kb of 5'franking region of cytochrome P-450 HP genome was cloned from human genomic library. Consensus sequences determined in other P-450 genome as a regulatory element were not found in the sequence. This result indicates that expression of cytochrome P-450 HP was regulated by unknown mechanism. Less
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