Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach.

Abstract:

:NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.

authors

Cui K,Lu AY,Yang CS

doi

10.1073/pnas.92.4.1043

subject

Has Abstract

pub_date

1995-02-14 00:00:00

pages

1043-7

issue

4

eissn

0027-8424

issn

1091-6490

journal_volume

92

pub_type

杂志文章
  • Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution.

    abstract::The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-A resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2(1) and contain a tetramer with 222 noncrystal...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.98.2.462

    authors: Yarbrough D,Wachter RM,Kallio K,Matz MV,Remington SJ

    更新日期:2001-01-16 00:00:00

  • Behavioral, neurochemical, and electrophysiological characterization of a genetic mouse model of depression.

    abstract::Depression is a multifactorial illness and genetic factors play a role in its etiology. The understanding of its physiopathology relies on the availability of experimental models potentially mimicking the disease. Here we describe a model built up by selective breeding of mice with strikingly different responses in th...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1034823100

    authors: El Yacoubi M,Bouali S,Popa D,Naudon L,Leroux-Nicollet I,Hamon M,Costentin J,Adrien J,Vaugeois JM

    更新日期:2003-05-13 00:00:00

  • Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum.

    abstract::Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohes...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1809283115

    authors: Barth A,Hendrix J,Fried D,Barak Y,Bayer EA,Lamb DC

    更新日期:2018-11-27 00:00:00

  • Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis.

    abstract::Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an alpha beta heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the alpha beta heterodimer, the b...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.84.14.5082

    authors: Hsueh AJ,Dahl KD,Vaughan J,Tucker E,Rivier J,Bardin CW,Vale W

    更新日期:1987-07-01 00:00:00

  • Proton transfer from the bulk to the bound ubiquinone Q(B) of the reaction center in chromatophores of Rhodobacter sphaeroides: retarded conveyance by neutral water.

    abstract::The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q(B) by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to Q(B) (at 450 nm), ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.96.23.13159

    authors: Gopta OA,Cherepanov DA,Junge W,Mulkidjanian AY

    更新日期:1999-11-09 00:00:00

  • Material-based deployment enhances efficacy of endothelial progenitor cells.

    abstract::Cell-based therapies are attractive for revascularizing and regenerating tissues and organs, but clinical trials of endothelial progenitor cell transplantation have not resulted in consistent benefit. We propose a different approach in which a material delivery system is used to create a depot of vascular progenitor c...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0803873105

    authors: Silva EA,Kim ES,Kong HJ,Mooney DJ

    更新日期:2008-09-23 00:00:00

  • Specificity in the activation and control of transcription factor E2F-dependent apoptosis.

    abstract::Previous work has demonstrated a role for the E2F1 gene product in signaling apoptosis, both as a result of the deregulation of the Rb/E2F pathway as well as in response to DNA damage. We now show that the ability of cells to suppress the apoptotic potential of E2F1, as might occur during the course of normal cellular...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1831408100

    authors: Hallstrom TC,Nevins JR

    更新日期:2003-09-16 00:00:00

  • Dynamics of enzymatic reactions.

    abstract::The detailed molecular dynamics of an actual bond-breaking event in a fluctuating enzyme substrate complex is simulated. The method developed allows one to explore what type of fluctuations are involved in enzymatic reactions and to evaluate entropic contributions to enzyme catalysis. The fluctuations of the enzyme el...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.81.2.444

    authors: Warshel A

    更新日期:1984-01-01 00:00:00

  • Protein-tyrosine-phosphatase CD45 is phosphorylated transiently on tyrosine upon activation of Jurkat T cells.

    abstract::The leukocyte common antigen (CD45) is an abundant lymphocyte surface antigen that has been reported to be involved in signaling through the T-cell antigen receptor. CD45 is a transmembrane protein-tyrosine-phosphatase. An internal segment comprises two domains each of which is homologous to other protein-tyrosine-pho...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.88.17.7704

    authors: Stover DR,Charbonneau H,Tonks NK,Walsh KA

    更新日期:1991-09-01 00:00:00

  • The Saccharomyces cerevisiae linker histone Hho1p is essential for chromatin compaction in stationary phase and is displaced by transcription.

    abstract::The importance of core histones in the regulation of DNA function by chromatin is clear. However, little is known about the role of the linker histone. We investigated the role of H1 in Saccharomyces cerevisiae during extensive transcriptional reprogramming in stationary phase. Although the levels of linker histone Hh...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0806337105

    authors: Schäfer G,McEvoy CR,Patterton HG

    更新日期:2008-09-30 00:00:00

  • Hormonal modulation of a gene injected into rat heart in vivo.

    abstract::We demonstrate gene transfer into rat heart in vivo by the direct injection of plasmid DNA. Injection of gene constructs driven by retroviral and cellular promoters resulted in detectable levels of reporter gene activities. The cellular promoter and 5' flanking sequence (positions -613 to +32) were derived from the ra...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.88.10.4138

    authors: Kitsis RN,Buttrick PM,McNally EM,Kaplan ML,Leinwand LA

    更新日期:1991-05-15 00:00:00

  • The molecular basis for the high photosensitivity of rhodopsin.

    abstract::Based on structural information derived from the F NMR data of labeled rhodopsins, rhodopsin crystal structure, and excited-state properties of model polyenes, we propose a molecular mechanism that accounts specifically for the causes of the well-known enhanced photoreactivity of rhodopsin (increased rates and quantum...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.2536769100

    authors: Liu RS,Colmenares LU

    更新日期:2003-12-09 00:00:00

  • Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

    abstract::Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-f...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1312457110

    authors: Liao SF,Liang CH,Ho MY,Hsu TL,Tsai TI,Hsieh YS,Tsai CM,Li ST,Cheng YY,Tsao SM,Lin TY,Lin ZY,Yang WB,Ren CT,Lin KI,Khoo KH,Lin CH,Hsu HY,Wu CY,Wong CH

    更新日期:2013-08-20 00:00:00

  • Metabolic hypothesis for human altriciality.

    abstract::The classic anthropological hypothesis known as the "obstetrical dilemma" is a well-known explanation for human altriciality, a condition that has significant implications for human social and behavioral evolution. The hypothesis holds that antagonistic selection for a large neonatal brain and a narrow, bipedal-adapte...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1205282109

    authors: Dunsworth HM,Warrener AG,Deacon T,Ellison PT,Pontzer H

    更新日期:2012-09-18 00:00:00

  • A shared cis-regulatory module activates transcription in the suspensor of plant embryos.

    abstract::The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean e...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1805802115

    authors: Henry KF,Bui AQ,Kawashima T,Goldberg RB

    更新日期:2018-06-19 00:00:00

  • Integration of stress-related and reactive oxygen species-mediated signals by Topoisomerase VI in Arabidopsis thaliana.

    abstract::Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen ((1)O(2)) in chloroplasts of the flu mutant of Arabidopsis, reprogramming of nuclear gene expression reveals a rapid transfer of ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1202041109

    authors: Simková K,Moreau F,Pawlak P,Vriet C,Baruah A,Alexandre C,Hennig L,Apel K,Laloi C

    更新日期:2012-10-02 00:00:00

  • Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis.

    abstract::Using a genome-wide approach, we asked how many transporter genes contribute to symbiotic phosphate uptake and analyzed their evolutionary conservation. Considering the sequenced rice genome at hand, only the Oryza sativa phosphate transporter (OsPT) gene OsPT11 was specifically induced during the arbuscular mycorrhiz...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.202474599

    authors: Paszkowski U,Kroken S,Roux C,Briggs SP

    更新日期:2002-10-01 00:00:00

  • Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon.

    abstract::Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon. Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling tech...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.82.14.4758

    authors: Kuchino Y,Hanyu N,Tashiro F,Nishimura S

    更新日期:1985-07-01 00:00:00

  • Benzodiazepine/gamma-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil.

    abstract::The density of benzodiazepine/gamma-aminobutyric acid receptor binding sites was lower in the midbrain of seizure-susceptible gerbils compared to control seizure-resistant gerbils. Binding of [3H]diazepam to high-affinity brain-specific sites in membrane homogenates of gerbil brain showed a 20-30% lower binding in mid...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.82.19.6701

    authors: Olsen RW,Wamsley JK,McCabe RT,Lee RJ,Lomax P

    更新日期:1985-10-01 00:00:00

  • CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.

    abstract::Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1700622114

    authors: Vanoli F,Tomishima M,Feng W,Lamribet K,Babin L,Brunet E,Jasin M

    更新日期:2017-04-04 00:00:00

  • Reversible inhibition of the ClpP protease via an N-terminal conformational switch.

    abstract::Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinoly...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1805125115

    authors: Vahidi S,Ripstein ZA,Bonomi M,Yuwen T,Mabanglo MF,Juravsky JB,Rizzolo K,Velyvis A,Houry WA,Vendruscolo M,Rubinstein JL,Kay LE

    更新日期:2018-07-10 00:00:00

  • Selective loss of wheat germ agglutinin binding to agglutinin-resistant mutants of Chinese hamster ovary cells.

    abstract::The binding of 125I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 10(6)-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was comple...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.74.11.5056

    authors: Stanley P,Carver JP

    更新日期:1977-11-01 00:00:00

  • Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation.

    abstract::Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1300035110

    authors: Niu F,Shaw N,Wang YE,Jiao L,Ding W,Li X,Zhu P,Upur H,Ouyang S,Cheng G,Liu ZJ

    更新日期:2013-05-28 00:00:00

  • Theorems of Barth-Lefschetz type for complex subspaces of homogeneous complex manifolds.

    abstract::Barth, Larsen, and others showed that complex submanifolds of complex projective space, CP(N), of small codimension strongly resemble CP(N) both homotopically and cohomologically. These results are generalized to yield analogous results for complex subspaces of arbitrary homogeneous complex manifolds. One very special...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.74.4.1332

    authors: Sommese AJ

    更新日期:1977-04-01 00:00:00

  • The primary step of biotin synthesis in mycobacteria.

    abstract::Biotin plays an essential role in growth of mycobacteria. Synthesis of the cofactor is essential for Mycobacterium tuberculosis to establish and maintain chronic infections in a murine model of tuberculosis. Although the late steps of mycobacterial biotin synthesis, assembly of the heterocyclic rings, are thought to f...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.2010189117

    authors: Hu Z,Cronan JE

    更新日期:2020-09-22 00:00:00

  • Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein.

    abstract::For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.91.17.8200

    authors: Li C,Peoples RW,Weight FF

    更新日期:1994-08-16 00:00:00

  • Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

    abstract::The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellu...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1318639110

    authors: Genovesi LA,Ng CG,Davis MJ,Remke M,Taylor MD,Adams DJ,Rust AG,Ward JM,Ban KH,Jenkins NA,Copeland NG,Wainwright BJ

    更新日期:2013-11-12 00:00:00

  • Cats use hollow papillae to wick saliva into fur.

    abstract::The cat tongue is covered in sharp, rear-facing spines called papillae, the precise function of which is a mystery. In this combined experimental and theoretical study, we use high-speed film, grooming force measurements, and computed tomography (CT) scanning to elucidate the mechanism by which papillae are used to gr...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1809544115

    authors: Noel AC,Hu DL

    更新日期:2018-12-04 00:00:00

  • An evolutionary process that assembles phenotypes through space rather than through time.

    abstract::In classical evolutionary theory, traits evolve because they facilitate organismal survival and/or reproduction. We discuss a different type of evolutionary mechanism that relies upon differential dispersal. Traits that enhance rates of dispersal inevitably accumulate at expanding range edges, and assortative mating b...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1018989108

    authors: Shine R,Brown GP,Phillips BL

    更新日期:2011-04-05 00:00:00

  • Consequences of seafood mislabeling for marine populations and fisheries management.

    abstract::Over the past decade, seafood mislabeling has been increasingly documented, raising public concern over the identity, safety, and sustainability of seafood. Negative outcomes from seafood mislabeling are suspected to be substantial and pervasive as seafood is the world's most highly traded food commodity. Here we prov...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.2003741117

    authors: Kroetz K,Luque GM,Gephart JA,Jardine SL,Lee P,Chicojay Moore K,Cole C,Steinkruger A,Donlan CJ

    更新日期:2020-12-01 00:00:00