Abstract:
:Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Trimboli AJ,Quinn GB,Smith ET,Barber MJdoi
10.1006/abbi.1996.0289subject
Has Abstractpub_date
1996-07-01 00:00:00pages
117-26issue
1eissn
0003-9861issn
1096-0384pii
S0003-9861(96)90289-1journal_volume
331pub_type
杂志文章abstract::M. Kuwajima, C. B. Newgard, D. W. Foster, and J. D. McGarry (1986, J. Biol. Chem. 261, 8849-8853) have concluded that the reason postprandial hepatic glycogenesis occurs primarily from gluconeogenic precursors rather than glucose is because glucokinase activity is insufficient to support the observed rates of glycogen...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(87)90648-5
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abstract::alpha 1-Acid glycoprotein (alpha 1-AGP) is a glucocorticoid inducible gene product that is synthesized and secreted by certain rat hepatoma tissue culture (HTC) cell lines such as M1.54. Exposure to monensin, a Na+-K+ ionophore, causes a significant redistribution of alpha 1-AGP into two distinct fractions; immunoprec...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(86)90491-1
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abstract::Myelin basic protein is known to be released into the circulation following traumatic injuries or demyelination within the central nervous system, resulting in the generation of potentially immunogenic myelin basic protein material. In this investigation we have studied the binding of bovine and human myelin basic pro...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1998.0902
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abstract::To assess the indirect effects of snake venom metalloproteinases (SVMP) on host tissue local necrosis, we investigated the effect of the SVMP jararhagin on the gene expression profiles of human fibroblasts in vitro and mouse tissue in vivo. Two functional classes of up-regulated proteins, cell death and inflammatory d...
journal_title:Archives of biochemistry and biophysics
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更新日期:2005-09-01 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::Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by i...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90102-3
更新日期:1992-10-01 00:00:00
abstract::Peribacteroid membranes can be isolated in essentially pure form from 20-day lupin root nodules by osmotic shock of the purified membrane enclosed bacteroids. The ATPase (EC 3.6.1.3) associated with this membrane has an acid pH optimum (5.25) and is specific for ATP (Mg-ATP Km = 0.16 mM). The enzyme activity requires ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(88)90322-0
更新日期:1988-08-01 00:00:00
abstract::The reactive cellular products generated during the peroxidation of membrane lipids have been implicated as causative agents in a variety of degenerative diseases and aging. In particular, 4-hydroxynon-2-enal (4HNE) is among the most of the produced during lipid peroxidation. In humans and rodent species, the alpha 4 ...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.2001.2352
更新日期:2001-06-01 00:00:00
abstract::Glutathione S-transferases (GSTs) of Plasmodium parasites are potential targets for antimalarial drug and vaccine development. We investigated the equilibrium unfolding, functional activity regulation and stability characteristics of the unique GST of Plasmodium vivax (PvGST). Despite high sequence, structural, functi...
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更新日期:2009-07-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90060-a
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doi:10.1016/j.abb.2009.02.008
更新日期:2009-05-15 00:00:00
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pub_type: 杂志文章
doi:10.1016/j.abb.2005.09.008
更新日期:2005-11-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1995.9962
更新日期:1995-11-10 00:00:00
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pub_type: 杂志文章
doi:10.1016/j.abb.2012.08.001
更新日期:2012-12-01 00:00:00
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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
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(84)90536-8
更新日期:1984-07-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(86)90742-3
更新日期:1986-11-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1006/abbi.1997.0249
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:1988-11-01 00:00:00
abstract::Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1997.0182
更新日期:1997-08-01 00:00:00
abstract::Conversion of a steroid receptor complex from the 8 S to the 4S form results in new interactions between the steroid and the receptor and/or formation of new intra-protein bonds within the receptor molecule itself. These bonds must be broken before the steroid is released. In order to localize these newly formed inter...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:1991-02-01 00:00:00
abstract::Rats fed an ethanol-containing diet for 4 weeks showed a 3- to 5-fold increase over isocalorically pair-fed controls with respect to cytosolic NAD(P)H-quinone oxidoreductase (NQOR) (E.C.1.6.99.2) with both menadione and dichlorophenol-indophenol as substrates. Rates of NAD(P)H-dependent p-nitrosophenol (pNSP) reductio...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90510-4
更新日期:1992-06-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(91)90128-6
更新日期:1991-12-01 00:00:00
abstract::Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the intera...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.2001.2398
更新日期:2001-07-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2009.03.003
更新日期:2009-05-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(87)90386-9
更新日期:1987-11-01 00:00:00
abstract::The so-called 90,000-Da heat shock protein (hsp90) from chicken liver has been purified and physically characterized in the presence of high levels of the serine phosphatase inhibitor fluoride. The protein is an elongated dimer with a molecular weight of 160,000 and a frictional ratio of 1.6. On two-dimensional electr...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(88)90569-3
更新日期:1988-07-01 00:00:00
abstract::The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e cons...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.2001.2741
更新日期:2002-03-15 00:00:00