TY - JOUR
T1 - Characterization of mRNA species related to human liver cytochrome P-450 nifedipine oxidase and the regulation of catalytic activity
AU - Bork, R. W.
AU - Muto, T.
AU - Beaune, P. H.
AU - Srivastava, P. K.
AU - Lloyd, R. S.
AU - Guengerich, F. P.
PY - 1989
Y1 - 1989
N2 - P-450(NF) is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine. By screening a bacteriophage λgt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated. This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P.H., Umbenhauer, D.R., Bork, R.W., Lloyd, R.S., and Guengerich, F.P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8064-8068) and HLp (Molowa, D.T., Schuetz, E.G., Wrighton, S.A., Watkins, P.B., Kremers, P., Mendez-Picon, G., Parker, G.A., and Guzelian, P.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5311-5315). However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal. Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level. Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of ~10:1. The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450(NF) and nifedipine oxidase activity in individual human liver samples. In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA. We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity.
AB - P-450(NF) is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine. By screening a bacteriophage λgt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated. This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P.H., Umbenhauer, D.R., Bork, R.W., Lloyd, R.S., and Guengerich, F.P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8064-8068) and HLp (Molowa, D.T., Schuetz, E.G., Wrighton, S.A., Watkins, P.B., Kremers, P., Mendez-Picon, G., Parker, G.A., and Guzelian, P.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5311-5315). However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal. Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level. Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of ~10:1. The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450(NF) and nifedipine oxidase activity in individual human liver samples. In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA. We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity.
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M3 - Article
C2 - 2463251
AN - SCOPUS:0024588897
SN - 0021-9258
VL - 264
SP - 910
EP - 919
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -