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

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidor eductase (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 previous ly reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378) , that the 2 forms - the hydrophilic and hydrophobic forms - of the mo use liver quinone reductase have the same molecular weight. No amino a cid sequence differences were found by tandem mass spectral analyses o f 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 red uctase have the same primary sequences, and that any difference betwee n the 2 forms may be attributed to a labile posttranslational modifica tion. Analysis of the mouse quinone reductase cDNA revealed that the e nzyme is 273 amino acids long and has a sequence homologous to those o f rat and human quinone reductases. In this study, the mouse quinone r eductase cDNA was also ligated into a prokaryotic expression plasmid p KK233.2, 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 am ino acid sequence similar to those of the rat and human enzymes, the m ouse enzyme has a higher NAD(P)H-menadione reductase activity and is l ess sensitive to flavones and dicoumarol, 2 known inhibitors of the en zyme. The results would indicate that the regions in mouse quinone red uctase that contain amino acids different from the rat and human enzym es are critical for the binding of menadione, flavones, and dicoumarol .