Enzymatic Determination of Phospholipase D Activity with Choline Oxidase

Imamura, Shigeyuki; Horiuti, Yoshifumi

doi:10.1093/oxfordjournals.jbchem.a131960

Cited by 93 publications

(64 citation statements)

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“…Hydrolysis of the physiological substrate is commonly detected by a fluorimetric method consisting of two coupling enzymes: choline oxidase and horseradish peroxidase (HRP) (Fig. 2) 30 . In this assay, choline oxidase converts choline into betaine and hydrogen peroxide, which HRP in turn uses to oxidize homovanillic acid (HVA), causing dimerization of HVA, allowing to reach its fluorophoric state (Scheme 1) 30 .…”

Section: Type II Atx Inhibitors Obstruct Lipid Binding To the Hydrophmentioning

confidence: 99%

“…2) 30 . In this assay, choline oxidase converts choline into betaine and hydrogen peroxide, which HRP in turn uses to oxidize homovanillic acid (HVA), causing dimerization of HVA, allowing to reach its fluorophoric state (Scheme 1) 30 . This indirect fluorescencebased method is the only assay to measure ATX catalytic activity towards its physiological substrate.…”

Section: Type II Atx Inhibitors Obstruct Lipid Binding To the Hydrophmentioning

confidence: 99%

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Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Preprint

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Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the "pocket" and the "tunnel". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

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Section: Type II Atx Inhibitors Obstruct Lipid Binding To the Hydrophmentioning

confidence: 99%

Section: Type II Atx Inhibitors Obstruct Lipid Binding To the Hydrophmentioning

confidence: 99%

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Preprint

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“…PLD activity was determined by measuring the amount of choline released from PC following the colorimetric-enzymatic method developed by Imamura and Horiuti (1978). The reaction mixture (0.5 ml) was composed of 10 mM CaCl 2 , 2 mM PC emulsion in 0.2 M acetate buffer (pH 5.5) in the presence or in the absence of 0.6 mM oleic acid.…”

Section: Pld Activitymentioning

confidence: 99%

Cold stress affects H + -ATPase and phospholipase D activity in Arabidopsis

Muzi

Camoni

Visconti

et al. 2016

Plant Physiology and Biochemistry

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a b s t r a c tLow temperature is an environmental stress that greatly influences plant performance and distribution. Plants exposed to cold stress exhibit modifications of plasma membrane physical properties that can affect their functionality. Here it is reported the effect of low temperature exposure of Arabidopsis plants on the activity of phospholipase D and H þ -ATPase, the master enzyme located at the plasma membrane.The H þ -ATPase activity was differently affected, depending on the length of cold stress imposed. In particular, an exposure to 4 C for 6 h determined the strong inhibition of the H þ -ATPase activity, that correlates with a reduced association with the regulatory 14-3-3 proteins. A longer exposure first caused the full recovery of the enzymatic activity followed by a significant activation, in accordance with both the increased association with 14-3-3 proteins and induction of H þ -ATPase gene transcription. Different time lengths of cold stress treatment were also shown to strongly stimulate the phospholipase D activity and affect the phosphatidic acid levels of the plasma membranes. Our results suggest a functional correlation between the activity of phospholipase D and H þ -ATPase mediated by phosphatidic acid release during the cold stress response.

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“…Further reacylation of glycerophosphatidylcholine with palmitic anhydride and 4-Dimethylaminopyridine (DMAP) as the catalyst was carried out according to the methods described by Regen et al to form dipalmitoyl phosphatidylcholine. 37,38 A crude extract of phospholipase D was obtained from fresh savoy cabbage and purified by hydrophobic interaction chromatography using the methods described by Lambrecht et al 39 The activity of phospholipase D cabbage was determined using choline oxidase and peroxsidase as described by Imamura et al 40 with DPPC produced earlier as a substrate. The amount of protein measured was determined using the Bradford test.…”

Section: Methodsmentioning

confidence: 99%

Simple Thermodynamically-Derived Model for Predicting the Hydrolase and Transferase Activity of Phospholipase D in the Synthesis of Phosphatidylglycerol

2012

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Preparation of a single and pure phospholipid via transphosphatidylation has been a much sought after endeavor in the pharmaceutical and nonmedical industries. For this reason, phosphatidylglycerol, a lung surfactant, was produced from phosphatidylcholine with defined fatty acids, ie, dipalmitoyl phosphatidylcholine. Substrate type and concentration, enzyme source, and reaction temperature were investigated. Phospholipase D from two sources, ie, savoy cabbage, was purified in the authors' laboratory and a commercially available Streptomyces species was used for this study. The substrates used were glycerol, a polyhydric alcohol, and solketal, a monohydric form of glycerol. The progress of the reaction was monitored using thin layer chromatography, and synthesis with solketal, an unusual form of glycerol, was confirmed by liquid chromatography mass spectrometry. Surface response methodology used on four combinations of enzyme and substrate at various temperatures (30 °C-60 °C) and concentration (0.25-1 mM) revealed that yield and selectivity was temperature-driven and predictable. To validate further the thermodynamic attributes, a modified version of the Eyring equation was derived from selectivity and the Arhenius equation. These equations provide some useful insights into the difference in activation of enthalpy change(ΔΔH ++ ) and difference in activation of entropy change(ΔΔS ++ ). Plots of ln[PG]/[PA] versus 1/T gave good linear fits for these four combinations. In addition, a new thermodynamic parameter known as T PG = PA has emerged as a theoretical temperature for equivalent transferase and hydrolase activity.

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Enzymatic Determination of Phospholipase D Activity with Choline Oxidase

Cited by 93 publications

References 0 publications

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Cold stress affects H + -ATPase and phospholipase D activity in Arabidopsis

Simple Thermodynamically-Derived Model for Predicting the Hydrolase and Transferase Activity of Phospholipase D in the Synthesis of Phosphatidylglycerol

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