Mouse liver NAD(P)H: quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis

S. Chen, P. E. Clarke, P. A. Martino, P. S K Deng, C. H. Yeh, T. D. Lee, H. J. Prochaska, Paul Talalay

Research output: Contribution to journalArticle

Abstract

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986. J Biol Chem 261:1372-1378), that the 2 forms- the hydrophilic and hydrophobic forms-of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme. The results would indicate that the regions in mouse quinone reductase that contain amino acids different from the rat and human enzymes are critical for the binding of menadione, flavones, and dicoumarol.

Original languageEnglish (US)
Pages (from-to)1296-1304
Number of pages9
JournalProtein Science
Volume3
Issue number8
StatePublished - 1994

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NAD(P)H Dehydrogenase (Quinone)
Cloning
Protein Sequence Analysis
Enzyme activity
Tandem Mass Spectrometry
Liver
Escherichia coli
Mass spectrometry
Organism Cloning
Complementary DNA
Enzymes
Proteins
Amino Acids
Dicumarol
Flavones
Rats
Amino Acid Sequence
Quinone Reductases
Plasmids
Vitamin K 3

Keywords

  • amino acid sequence
  • cDNA
  • mouse liver quinone reductase
  • tandem mass spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mouse liver NAD(P)H : quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis. / Chen, S.; Clarke, P. E.; Martino, P. A.; Deng, P. S K; Yeh, C. H.; Lee, T. D.; Prochaska, H. J.; Talalay, Paul.

In: Protein Science, Vol. 3, No. 8, 1994, p. 1296-1304.

Research output: Contribution to journalArticle

Chen, S. ; Clarke, P. E. ; Martino, P. A. ; Deng, P. S K ; Yeh, C. H. ; Lee, T. D. ; Prochaska, H. J. ; Talalay, Paul. / Mouse liver NAD(P)H : quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis. In: Protein Science. 1994 ; Vol. 3, No. 8. pp. 1296-1304.
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abstract = "The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986. J Biol Chem 261:1372-1378), that the 2 forms- the hydrophilic and hydrophobic forms-of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme. The results would indicate that the regions in mouse quinone reductase that contain amino acids different from the rat and human enzymes are critical for the binding of menadione, flavones, and dicoumarol.",
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