Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase

Cecile Ged, Diane R. Umbenhauer, Teresa M. Bellew, Richard W. Bork, Pramod K. Srivastava, Nariko Shinriki, Robert (Stephen) Lloyd, F. Peter Guengerich

Research output: Contribution to journalArticle

122 Citations (Scopus)

Abstract

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4′-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using nonoverlapping 5′ and 3′ portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage λgt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4′-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3′ noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs - individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4′-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4′-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

Original languageEnglish (US)
Pages (from-to)6929-6940
Number of pages12
JournalBiochemistry
Volume27
Issue number18
StatePublished - 1988
Externally publishedYes

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Liver
Cytochrome P-450 Enzyme System
Mephenytoin
Complementary DNA
Clone Cells
Messenger RNA
Hydroxylation
Genes
Proteins
Catalyst activity
Bacteriophages
Oligonucleotide Probes
DNA sequences
Protein Sequence Analysis
Enzyme activity
Genetic Polymorphisms
Multigene Family
Southern Blotting
Mixed Function Oxygenases
Cytochrome P-450 CYP2C8

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ged, C., Umbenhauer, D. R., Bellew, T. M., Bork, R. W., Srivastava, P. K., Shinriki, N., ... Guengerich, F. P. (1988). Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase. Biochemistry, 27(18), 6929-6940.

Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase. / Ged, Cecile; Umbenhauer, Diane R.; Bellew, Teresa M.; Bork, Richard W.; Srivastava, Pramod K.; Shinriki, Nariko; Lloyd, Robert (Stephen); Guengerich, F. Peter.

In: Biochemistry, Vol. 27, No. 18, 1988, p. 6929-6940.

Research output: Contribution to journalArticle

Ged, C, Umbenhauer, DR, Bellew, TM, Bork, RW, Srivastava, PK, Shinriki, N, Lloyd, RS & Guengerich, FP 1988, 'Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase', Biochemistry, vol. 27, no. 18, pp. 6929-6940.
Ged C, Umbenhauer DR, Bellew TM, Bork RW, Srivastava PK, Shinriki N et al. Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase. Biochemistry. 1988;27(18):6929-6940.
Ged, Cecile ; Umbenhauer, Diane R. ; Bellew, Teresa M. ; Bork, Richard W. ; Srivastava, Pramod K. ; Shinriki, Nariko ; Lloyd, Robert (Stephen) ; Guengerich, F. Peter. / Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4′-hydroxylase. In: Biochemistry. 1988 ; Vol. 27, No. 18. pp. 6929-6940.
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abstract = "A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4′-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using nonoverlapping 5′ and 3′ portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage λgt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85{\%} identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4′-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3′ noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs - individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4′-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4′-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).",
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