Publications

• Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein.

  Tang A., Curthoys N.P.

  Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to ... >> More

  Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein. << Less

  J Biol Chem 276:21375-21380(2001) [PubMed] [EuropePMC]

• Kinetic properties of camel lens zeta-crystallin.

  Duhaiman A.S.

  Camel lens zeta-crystallin is an NADPH:quinone oxidoreductase, showing limited quinone substrate specificity: among the quinones tested, the orthoquinones were the best substrates. The kinetic mechanism of NADPH:quinone oxidoreductase activity of zeta-crystallin was investigated by steady-state in ... >> More

  Camel lens zeta-crystallin is an NADPH:quinone oxidoreductase, showing limited quinone substrate specificity: among the quinones tested, the orthoquinones were the best substrates. The kinetic mechanism of NADPH:quinone oxidoreductase activity of zeta-crystallin was investigated by steady-state initial velocity and product-inhibition studies. The results were consistent with the enzyme having a ping-pong mechanism and the intrinsic K(m) values for NADPH and 9,10-phenanthrenequinone (PQ) were estimated to be 48.0 +/- 2.5 and 36.0 +/-1.5 microM, respectively. NADP+ showed a mixed type of inhibition with respect to NADPH, while it was a competitive inhibitor with respect to PQ. Dithiothreitol (DTT) was a strong competitive inhibitor with respect to PQ with a Ki value of 50 microM. Similarly, 2, 3-dimercaptopropanol inhibited the enzyme activity with a Ki value of 260 microM. The enzyme was inactivated by sulfhydryl groups, modifying agents such as 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) and N-ethyl-maleimide. The substrate specificities and kinetic properties of camel lens NADPH:quinone oxidoreductase reported here distinguish it from previously characterized quinone oxidoreductases. This paper reports, for the first time, on the kinetic mechanism of camel lens zeta-crystallin. << Less

  Int J Biochem Cell Biol 28:1163-1168(1996) [PubMed] [EuropePMC]

• Interaction of camel lens zeta-crystallin with quinones: portrait of a substrate by fluorescence spectroscopy.

  Bazzi M.D.

  Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoqui ... >> More

  Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoquinone), 1,4 naphthoquinone (1,4-NQ), and 1,2 naphthoquinone (1,2-NQ) considerably quenched the fluorescence of zeta-crystallin, where as the commonly used substrate, 9,10-phenanthrenequinone (PQ) did not induce significant quenching. Activity measurements showed only PQ served as a substrate for camel lens zeta-crystallin, while juglone, 1,4-NQ, and 1,2-NQ were inhibitors. Thus quinones that interacted with zeta-crystallin directly inhibited the enzyme, whereas the substrate had very low affinity for the enzyme in the absence of NADPH. Another substrate, dichlorophenol indophenol (DCIP), conformed to the same pattern; DCIP did not quench the fluorescence of the enzyme significantly, but served as a substrate. This pattern is consistent with an ordered mechanism of catalysis with quinone being the second substrate. All three naphthoquinones were uncompetitive inhibitors with respect to NADPH and noncompetitive with respect to PQ. These kinetics are similar to those exhibited by cysteine-and/or lysine-modifying agents. Juglone, 1,4-NQ, and 1,2-NQ interacted with and quenched the fluorescence of camel lens alpha-crystallin, but to lesser extent than that of zeta-crystallin. << Less

  Arch Biochem Biophys 395:185-190(2001) [PubMed] [EuropePMC]

• Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

  Rao P.V., Krishna C.M., Zigler J.S. Jr.

  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 limit ... >> More

  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. << Less

  J. Biol. Chem. 267:96-102(1992) [PubMed] [EuropePMC]