A Robotics-Based Automated Assay for Inorganic and Organic Phosphates

Cogan, Elizabeth B.; Birrell, G. Bruce; Griffith, O. Hayes

doi:10.1006/abio.1999.4100

Cited by 160 publications

(107 citation statements)

References 34 publications

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“…Quantification of TAP-associated Lipids-After purification, lipids bound to TAP were determined by acidic digestion and subsequent colorimetric detection of free phosphate via ammonium molybdate (28,29) Lipid Analysis by Mass Spectrometry-Lipids were extracted by chloroform/methanol (2:1, v/v) and analyzed by normal phase liquid chromatography using a PVA-SIL column (YMC Europe GmbH) interfaced with a nanoflow ion source Triversa NanoMate (Advion Biosciences, Inc.) and a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). PC, PE, and PI species were monitored in positive ion mode by recording FT-MS spectra with a target mass resolution of 100,000.…”

Section: Methodsmentioning

confidence: 99%

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

Schölz

Parcej

Ejsing

et al. 2011

Journal of Biological Chemistry

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The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.

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Section: Methodsmentioning

confidence: 99%

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

Schölz

Parcej

Ejsing

et al. 2011

Journal of Biological Chemistry

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“…The concentration of GPE was estimated by calculating the total phosphate concentration after total acid hydrolysis, performed according to Kates (23). Phosphate concentration was estimated by the phosphate detection assay of Cogan et al (9). The purity of the substrates was Ͼ95%, with the exception of GPE, which contained ϳ10% inorganic phosphate.…”

Section: Methodsmentioning

confidence: 99%

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

McLoughlin

Jackson

Liu

et al. 2004

Appl Environ Microbiol

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Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.Organophosphates (OPs) have been used as pesticides and chemical warfare agents during the last 60 years. OPs are considered nonpersistent compounds, breaking down in the environment over time to form relatively nontoxic compounds due to the action of light, water, and soilborne organisms. The bioremediation of sites contaminated with OPs is of concern in circumstances where there is a high risk of contact with humans. Escherichia coli, Moraxella sp., and Pseudomonas putida expressing recombinant bacterial phosphotriesterases (PTEs) have been used to detoxify solutions of phosphotriester pesticides (24, 34, 47) and could potentially be used for the bioremediation of contaminated soil and water. PTEs have been characterized with high activity towards particular OPs (e.g., paraoxon), but relatively low activities towards other OPs (e.g., demeton) (12,20,29). For the bioremediation of sites contaminated with poor PTE substrates, it is desirable to evolve PTEs with enhanced activity towards such substrates.Several groups have undertaken the directed evolution of PTEs for higher activity towards poor substrates (7,8,49). The major difficulty in generating such an enzyme is effectively screening large mutant libraries for mutants with higher activity. To this end, we are investigating the possibility of coupling PTE activity with cell growth that uses a phosphotriester as the sole phosphorus source. The enzyme expression leve...

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“…ATPase Assay-This assay was based on a modification of a published procedure (12). The DNA template (50-mer/70-mer complementary synthetic oligonucleotides) was mixed with DnaX complex and ␤ (20 l) in ATPase reaction buffer.…”

Section: Reconstitution Of Dnax Complexes Containing Mixtures Of Wildmentioning

confidence: 99%

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

Wieczorek

Downey

Dallmann

et al. 2010

Journal of Biological Chemistry

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The DnaX complex (DnaX 3 ␦␦) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, ␤ 2 , onto DNA. The complex contains three DnaX subunits, which occur in two forms: and the shorter ␥, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant or ␥ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATPbinding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. ␤ 2 binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for ␤ 2 . DNA synthesis activity can be restored by increased concentrations of ␤ 2 . In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances ␤ 2 loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes. DNA polymerase III holoenzyme (pol III HE)2 exhibits features common to all other cellular replicases. It is a tripartite assembly composed of a replicative polymerase (pol III), a sliding clamp processivity factor (␤ 2 ), and an AAAϩ ATPase that assembles the ␤ 2 clamp around DNA (DnaX complex) (1, 2). The DnaX complex contains three DnaX subunits and one each of ␦, ␦Ј, , and . The DnaX subunit is either the full-length dnaX translation product or a shorter version, ␥, which is generated by translational frameshifting. ␥ lacks two C-terminal domains that bind the replicative helicase and pol III.Current models for clamp loading, largely derived from ␥-only DnaX complexes in the absence of pol III, propose that ATP binds to all three DnaX subunits, increasing the affinity of the clamp loader for ␤ 2 and primed DNA (1, 3). Once a ␤ 2 -primed DNA complex is formed, hydrolysis of the three ATPs places the DnaX complex into a conformation with decreased affinity for DNA, leading to its dissociation from DNA-bound ␤ 2 (3). pol III then associates with ␤ 2 in a downstream step, leading to formation of an initiation complex for processive replication.We have shown that some of the characteristics of initiation complex formation catalyzed by -containing DnaX complexes are different from ␥-only complexes. For example, complex, when bound to pol III, can form initiation complexes in the leading strand half of a dimeric replicase in the presence of the nonhydrolyzed an...

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A Robotics-Based Automated Assay for Inorganic and Organic Phosphates

Cited by 160 publications

References 34 publications

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

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