A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-d-erythritol 4-phosphate synthase activity

Bernal, Cristobal; Palacín, Creu; Boronat, Albert; Imperial, Santıago

doi:10.1016/j.ab.2004.10.011

Cited by 37 publications

(29 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1A showed the absorption spectra of standard sodium dihydrogen phosphate, in complex with dye reagent mentioned above, from the wavelength of 550e750 nm. The spectra exhibited a characteristic absorption peak approximately at 650 nm, which deviated slightly from previously proposed 620 nm [18,19] and 630 nm [20]. Then the standard curve was determined in the 0e300 mM range of sodium dihydrogen phosphate (Fig.…”

Section: Optimization Of the Parameters Of Malachite Green Methodsmentioning

confidence: 70%

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Yang

Wang

2016

Analytical Biochemistry

View full text Add to dashboard Cite

Section: Optimization Of the Parameters Of Malachite Green Methodsmentioning

confidence: 70%

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Yang

Wang

2016

Analytical Biochemistry

View full text Add to dashboard Cite

“…Detection of free phosphates was performed as previously described (Bernal et al, 2005;Moeder et al, 2013).…”

Section: Malachite Green Assaymentioning

confidence: 99%

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

et al. 2017

View full text Add to dashboard Cite

ORCID IDs: 0000-0003-3889-6183 (W.M.); 0000-0002-3797-4277 (K.Y.).The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

show abstract

“…Free phosphate released by AtTTM2 was measured with the Malachite Green assay (Bernal et al, 2005) as described by Moeder et al (2013). The assay conditions were 0.5 mM PPi, ATP, or PPPi and 2.5 mM Mg 2+ , pH 9.0 at 37°C for 30 min.…”

Section: Enzymatic Assaysmentioning

confidence: 99%

Arabidopsis Triphosphate Tunnel Metalloenzyme2 Is a Negative Regulator of the Salicylic Acid-Mediated Feedback Amplification Loop for Defense Responses

2014

View full text Add to dashboard Cite

The triphosphate tunnel metalloenzyme (TTM) superfamily represents a group of enzymes that is characterized by their ability to hydrolyze a range of tripolyphosphate substrates. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes, AtTTM1, AtTTM2, and AtTTM3. Although AtTTM3 has previously been reported to have tripolyphosphatase activity, recombinantly expressed AtTTM2 unexpectedly exhibited pyrophosphatase activity. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. In addition, stronger systemic acquired resistance compared with wild-type plants was observed. These enhanced defense responses are dependent on SA, PHYTOALEXIN-DEFICIENT4, and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. Despite their enhanced pathogen resistance, ttm2 plants did not display constitutively active defense responses, suggesting that AtTTM2 is not a conventional negative regulator but a negative regulator of the amplification of defense responses. The transcriptional suppression of AtTTM2 by pathogen infection or treatment with SA or the systemic acquired resistance activator benzothiadiazole further supports this notion. Such transcriptional regulation is conserved among TTM2 orthologs in the crop plants soybean (Glycine max) and canola (Brassica napus), suggesting that TTM2 is involved in immunity in a wide variety of plant species. This indicates the possible usage of TTM2 knockout mutants for agricultural applications to generate pathogen-resistant crop plants.

show abstract

A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-d-erythritol 4-phosphate synthase activity

Cited by 37 publications

References 37 publications

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

Arabidopsis Triphosphate Tunnel Metalloenzyme2 Is a Negative Regulator of the Salicylic Acid-Mediated Feedback Amplification Loop for Defense Responses

Contact Info

Product

Resources

About