The Use of Protocatechuate Dioxygenase for Maintaining Anaerobic Conditions in Biochemical Experiments

Patil, Pravin; Ballou, David P.

doi:10.1006/abio.2000.4802

Cited by 99 publications

(99 citation statements)

References 5 publications

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“…We found that both galactose oxidase and protocatechuate dioxygenase (PCD) are compatible with splicing and both are as effective as glucose oxidase in extending fluorophore lifetimes. Indeed, of the three enzyme systems, PCD may be best suited for SMF because the enzyme is remarkably stable (Patil and Ballou 2000), does not contain a fluorescent (flavin) cofactor, and does not generate H 2 O 2 .…”

Section: Discussionmentioning

confidence: 99%

“…1a;Patil and Ballou 2000). Bulk assays indicated that addition of Pichia pastoris galactose oxidase (Whittaker and Whittaker 2000), bovine liver catalase, Pseudomonas PCD, Burkholderia cepacia PCD (Patil and Ballou 2000), 30 mM galactose, and/or 5 mM protocatechuate had no inhibitory effects on splicing of the well-characterized RP51A pre-mRNA transcript (Fig.…”

Section: Single-molecule Fluorescence In Cell Extractsmentioning

confidence: 93%

“…Activated (green form) P. pastoris galactose oxidase (1400 U/mg, 10 mg/mL) (Whittaker and Whittaker 2000), a generous gift from James Whittaker (Oregon Health and Sciences University), was added to a final concentration of 140 U/mL, along with 30 mM galactose and 1500 U/mL catalase. B. cepacia PCD (5 U/mg, 9 mg/mL), a generous gift of David Ballou (University of Michigan), was assayed before use (Patil and Ballou 2000) and added to a final concentration of 0.9 U/mL along with 5 mM protocatechuate. Protocatechuate (Sigma) was recrystallized twice from hot water before use.…”

Section: Oxygen Scavengingmentioning

confidence: 99%

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Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Crawford¹,

Hoskins²,

Friedman³

et al. 2007

RNA

114

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The excision of introns from nascent eukaryotic transcripts is catalyzed by the spliceosome, a highly complex and dynamic macromolecular machine composed of RNA and protein. Because of its complexity, biochemical analysis of the spliceosome has been previously limited to bulk assays in largely unfractionated cell extracts. We now report development of methodologies for studying the splicing of isolated single pre-mRNA molecules in real time. In this system, a fluorescently tagged pre-mRNA is tethered to a glass surface via its 39-end. Splicing can be observed in Saccharomyces cerevisiae whole cell extract by monitoring loss of intron-specific fluorescence with a multi-wavelength total internal reflection fluorescence (TIRF) microscope. To prolong fluorophore lifetime, two enzyme-based O 2 scavenging systems compatible with splicing were also developed. This work provides a powerful new approach for elucidating the mechanisms of spliceosome function and demonstrates the feasibility of utilizing TIRF microscopy for biochemical studies of single molecules in highly complex environments.

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Section: Discussionmentioning

confidence: 99%

Section: Single-molecule Fluorescence In Cell Extractsmentioning

confidence: 93%

Section: Oxygen Scavengingmentioning

confidence: 99%

See 1 more Smart Citation

Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Crawford¹,

Hoskins²,

Friedman³

et al. 2007

RNA

114

View full text Add to dashboard Cite

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“…For anaerobic reactions, the system was flushed and incubated overnight with a solution containing 3,4-dihydroxybenzoate (protocatechuic acid) and protocatechuate 3,4-dioxygenase to scavenge any oxygen (21). All buffers and Ph 2 I ϩ solutions were made anaerobic by bubbling with highly pure argon for 15 min in a syringe before use.…”

Section: Methodsmentioning

confidence: 99%

Reaction of Reduced Flavins and Flavoproteins with Diphenyliodonium Chloride

Chakraborty

Massey²

2002

Journal of Biological Chemistry

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The reaction of diphenyliodonium chloride with free reduced flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained ϳ20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993) Biochemistry 32, 10209 -10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.

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“…Solutions were repeatedly evacuated and flashed with purified nitrogen. Anaerobic samples included an oxygen scrubbing system of 100 -200 M protocatechuic acid and 0.02 units/ml protocatechuate dioxygenase (8). Concentrations of WT Pdr were calculated using an extinction coefficient of 10.4 mM Ϫ1 cm Ϫ1 at 455 nm (2).…”

Section: Methodsmentioning

confidence: 99%

Putidaredoxin Reductase, a New Function for an Old Protein

Sevrioukova¹,

Poulos²

2002

Journal of Biological Chemistry

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Properties of recombinant wild type (WT) and sixhistidine tag-fused (His 6 ) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied. Both WT and His 6 Pdr were found to undergo a monomer-dimer associationdissociation and were partially present as an NAD ؉ -bound form. Although molecular, spectral, and electron transferring properties of recombinant His 6 Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-HisHis-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide. Under anaerobic conditions, NAD ؉ induced in His 6 Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD ؉ . The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione. In the presence of any of these three reagents, NAD ؉ was capable of inducing reduction of the flavin in WT Pdr. Free thiol groups were identified as an internal source of electrons in the enzyme. The results showed that WT and His 6 Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD ؉ -dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin. Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.A FAD-containing putidaredoxin reductase (Pdr) 1 catalyzes the transfer of electrons from NADH to an iron-sulfur protein, putidaredoxin (Pdx), in the cytochrome P450cam monooxygenase system from Pseudomonas putida (1). Two-electron reduction of FAD with NADH is followed by the transfer of two single electrons from the flavin to Pdx. From Pdx, electrons are finally transferred to the terminal oxygenase cytochrome P450cam (CYP101) that catalyzes hydroxylation of camphor consuming two electrons and molecular oxygen per reaction cycle.Pdr has a molecular mass of 45.6 kDa and contains one tightly bound FAD per single polypeptide. Redox measurements, where sodium dithionite was used as a reductant, demonstrated that the midpoint potential of Pdr is Ϫ285 mV (2). The reduction of Pdr by NADH is a stoichiometric process that results in the formation of a long-wavelength charge transfer complex without the appearance of a semiquinone intermediate (3). The reduction of Pdr with NADPH is approximately 3 orders of magnitude slower than with NADH (4). No charge transfer band could be detected when the flavoprotein was reduced with either NADPH or sodium dithionite. Using NADPH as a reductant, the midpoint potential of Pdr was calculated to be Ϫ315 mV (4). Titration of Pdr with NADPH in the presence of excess NAD ϩ resulted in a stoichiometric reduction. This was explained as ...

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The Use of Protocatechuate Dioxygenase for Maintaining Anaerobic Conditions in Biochemical Experiments

Cited by 99 publications

References 5 publications

Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Reaction of Reduced Flavins and Flavoproteins with Diphenyliodonium Chloride

Putidaredoxin Reductase, a New Function for an Old Protein

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