Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.
zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.
Rao
P V
PV
Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Krishna
C M
CM
Zigler
J S
JS
D000818:Animals; D002384:Catalysis; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006168:Guinea Pigs; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009285:Naphthoquinones / Q000378:metabolism; D009582:Nitrofurantoin / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism; D013113:Spin Labels; D013379:Substrate Specificity
D003459:Crystallins; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D009285:Naphthoquinones; D011809:Quinones; D013113:Spin Labels; D009249:NADP; D001728:Dicumarol; D009582:Nitrofurantoin; D006861:Hydrogen Peroxide; D011808:Quinone Reductases; D010100:Oxygen; C001803:tempol; C005134:juglone