Abstract:
:Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Roma GW,Crowley LJ,Barber MJdoi
10.1016/j.abb.2006.04.021subject
Has Abstractpub_date
2006-08-01 00:00:00pages
69-82issue
1eissn
0003-9861issn
1096-0384pii
S0003-9861(06)00167-6journal_volume
452pub_type
杂志文章abstract::Mitochondrial carbonic anhydrase has previously been quantitated in liver mitochondria; it was not detected in guinea pig kidney cortical mitochondria. Evidence of this enzyme in rat kidney cortical mitochondria is reported. Electron microscopy showed that intact mitochondria were free of other intracellular organelle...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(88)90457-2
更新日期:1988-01-01 00:00:00
abstract::The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphog...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(90)90526-5
更新日期:1990-07-01 00:00:00
abstract:BACKGROUND:The main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol,...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2019.03.005
更新日期:2019-05-15 00:00:00
abstract::We have characterized a Brassica rapa mutant (WR586) that has low levels of polyunsaturated fatty acids (D.L. Auld et al., 1992, Crop Sci. 32, 657-662). The mutant lacked oleoyl-phosphatidylcholine desaturase (ODS) activity when assayed in 6-day-old seedlings. To further characterize the mutant, the leaf fatty acid co...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1994.1491
更新日期:1994-11-15 00:00:00
abstract::We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2015.02.021
更新日期:2015-03-15 00:00:00
abstract::The effect of a protein matrix on the processing of glycoprotein glycans by Golgi enzymes from plant seedlings has been determined with an artificial glycoprotein system, comparing the processing rates of glycan-(biotinyl)Asn (or glycan-(biotinamidohexanoyl)Asn) substrates either free or bound to avidin. An analysis o...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(91)90020-j
更新日期:1991-04-01 00:00:00
abstract::Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirme...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1998.0926
更新日期:1998-12-01 00:00:00
abstract::MicroRNA-342-3p (miR-342) has been shown to act as a tumor-suppressor in different cancer types. However, the role and therapeutic implications of miR-342 via modulation of Cofilin 1 (CFL1) has not been studied in any type of cancer. Given the importance of Cofilin signalling in breast, this study was undertaken to ex...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2020.108385
更新日期:2020-07-15 00:00:00
abstract::Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2012.03.019
更新日期:2012-05-01 00:00:00
abstract::Calorie restriction (CR) has been shown to improve peripheral insulin resistance and type 2 diabetes in animal models. However, the exact mechanism of CR on GLUT4 expression and translocation in insulin-sensitive tissues has not been well elucidated. In the present study, we examine the effect of CR on the expression ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2005.01.010
更新日期:2005-04-15 00:00:00
abstract::Flow cytometry was used to test the role of the propeptide of procathepsin D in the activation of human peripheral lymphocytes and neutrophils. A selective inhibition of the activation was achieved by antibodies targeted against the propeptide. Synthetic peptide corresponding to the cathepsin D propeptide had a very s...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1995.1466
更新日期:1995-09-10 00:00:00
abstract::The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible "hinge". While this region has not been structurally resolved, it likely acts as an extended linker between ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2014.01.016
更新日期:2014-06-15 00:00:00
abstract::Stark absorption spectroscopy was applied to clarify the structural differences between carotenoids bound to the B800-820 and B800-850 LH2 complexes from a purple photosynthetic bacterium Phaeospirillum (Phs.) molischianum DSM120. The former complex is produced when the bacteria are grown under stressed conditions of ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2014.12.015
更新日期:2015-04-15 00:00:00
abstract::The formation of alpha-hydroxyethyl radical from ethanol by deermouse microsomes supplemented with NADPH has been demonstrated with the EPR technique of spin trapping with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) as the spin trap, in the presence of deferoxamine mesylate. Superoxide dismutase prevented the fo...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1993.1293
更新日期:1993-06-01 00:00:00
abstract::A kinetic study of acetaminophen oxidation by tyrosinase in the presence of a physiological nucleophilic agent such as the amino acid L-proline is performed in the present paper. The o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, becomes unstable through the chemical addition of L-proline, in...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/s0003-9861(03)00288-1
更新日期:2003-08-15 00:00:00
abstract::The reduction of duroquinone (DQ), 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), and dichlorophenol indophenol (DCIP) by succinate and NADH was investigated in yeast mitochondria which have no spectrally detectable cytochrome b. Succinate reduces DB in the cytochrome b-deficient mitochondria at rates comparabl...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(88)90148-8
更新日期:1988-09-01 00:00:00
abstract::Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin. Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly co...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90749-m
更新日期:1992-05-01 00:00:00
abstract::The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1997.0444
更新日期:1998-01-15 00:00:00
abstract::Heparin is thought to regulate the rate of mammalian blood clotting by enhancing the activity of antithrombin, an inhibitor of coagulation enzymes. The present study establishes that this same inhibitor is present in the blood plasma of each of the terrestrial vertebrate groups including mammals, birds, reptiles, and ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(83)90488-5
更新日期:1983-12-01 00:00:00
abstract::The Arabidopsis thaliana genome contains at least 32 terpenoid synthase (AtTPS) genes [Aubourg et al., Mol. Genet. Genom. 267 (2002) 730] a few of which have recently been characterized. Based on hierarchical cluster analysis of AtTPS gene expression, measured by microarray profiling and validated with published expre...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2005.09.019
更新日期:2006-04-15 00:00:00
abstract::The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.2001.2390
更新日期:2001-07-01 00:00:00
abstract::Escherichia coli pyruvate oxidase (POXEC) requires FAD both for the oxidative decarboxylation of pyruvate to acetate and CO2 and for the formation of acetoin from pyruvate and acetaldehyde. Prior work has shown that the catalytic activity (kcat/Km) for POXEC in the oxidative reaction is stimulated approximately 450-fo...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1993.1049
更新日期:1993-01-01 00:00:00
abstract::The epithelial cells of the gastrointestinal tract have the capacity to engage in biotransformation of ingested chemicals. A principal component of phase I metabolism of xenobiotics is the family of hemeproteins referred to as cytochrome(s) P450. The presence of cytochrome P450 isoforms was examined by Western blot an...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(91)90288-t
更新日期:1991-01-01 00:00:00
abstract::The didecameric hemocyanin of the bivalve mollusc Nucula hanleyi, observed in the electron microscope in vitreous ice, was subjected to a three-dimensional (3D) reconstruction by the random conical tilt series method. The 3D volume was analyzed by computing solid-body surface representations and slices through the vol...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1995.1287
更新日期:1995-05-10 00:00:00
abstract::Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90703-y
更新日期:1992-05-01 00:00:00
abstract::For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (Kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate co...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(86)90445-5
更新日期:1986-04-01 00:00:00
abstract::The cross-reactivity of the monoclonal anti-human placental DNA methyltransferase antibody M2B10 with DNA methyltransferases isolated from other species was investigated. This antibody immunoprecipitates DNA methyltransferases from mammalian cells, i.e., human placenta, mouse P815 cells, and rat liver cells. No cross-...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(89)90599-7
更新日期:1989-01-01 00:00:00
abstract::Dipeptidyl-peptidase IV (EC 3.4.14.5) also known as CD26 is a membrane-bound serine peptidase which cleaves N-terminal dipeptides from a peptide chain containing a proline residue in the penultimate position. The enzyme is believed to play an important role in neuropeptide metabolism and T-cell activation. A series of...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1995.0020
更新日期:1995-10-20 00:00:00
abstract::The Metabotropic glutamate receptor 2 (mGluR2) is involved in several neurological and psychiatric disorders and is an attractive drug target. It is believed to form a strict dimer and the dimeric assembly is necessary for glutamate induced activation. Although many studies have focused on glutamate induced conformati...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2020.108632
更新日期:2021-01-15 00:00:00
abstract::Hydrogen peroxide triggers a redox cycle between methemoglobin and ferrylhemoglobin, leading to protein inactivation and oxygen evolution. In the present paper, the catalase-like oxygen production by human methemoglobin in the presence of H(2)O(2) was kinetically characterized with a Clark-type electrode. Progress cur...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2011.09.006
更新日期:2011-12-01 00:00:00