A coupled spectrophotometric assay for trehalose 6-phosphate synthetase from Dictyostelium discoideum

Killick, Kathleen A.

doi:10.1016/s0147-5975(77)80020-0

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…The classical assay for the detection and determination of TPS activity is a pyruvate kinase/lactate dehydrogenase‐coupled assay measuring the TPS‐induced production of UDP via the spectrophotometric detection of dihydronicotineamide adenine dinucleotide (NADH) oxidation (Killick, 1977b,Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…The classical TPS assays used radioactively labelled UDP‐glucose or glucose‐6‐phosphate substrates and required complex downstream processing, which is clearly not ideal for adaptation to HTS (Candy & Kilby, 1959, 1961; Murphy & Wyatt, 1965; Killick, 1977a). A coupled spectrophotometric assay using PK and lactate dehydrogenase (LDH) to detect via NADH oxidation the UDP generated by the TPS reaction has been developed (Killick, 1977b), but the low dynamic range of NADH consumption measurement and the problem of stopping the enzyme reaction without interfering with NADH/NAD + led us to seek novel alternative assay formats. The most robust solution comprised the omission of the LDH reaction and the detection of PK‐generated pyruvate via fluorogenic hydrazone formation with NBD hydrazine.…”

Section: Discussionmentioning

confidence: 99%

“…Column eluates were analysed by SDS‐PAGE and Coomassie blue staining. Detection of TPS enzyme activity in column fractions was performed by a coupled spectrophotometric assay (Killick, 1977b) in a buffer containing 50 mM Tris‐HCl pH 7.5, 100 mM KCl, 2.5 mM glucose‐6‐phosphate, 2.5 mM UDP‐glucose, 2.5 mM PEP, 0.2 mM NADH, 2 U/ml pyruvate kinase, 2 U/ml lactate dehydrogenase. Column fractions were added and the decrease in OD at 340 nm was recorded.…”

Section: Methodsmentioning

confidence: 99%

“…For HTS of MalE-DmTPS, the NADH/NAD + readout of the TPS assay (Killick, 1977b) needed to be transformed into a novel format with a larger dynamic range and more amenable to endpoint measurement. This was achieved by replacing the LDH reaction for the detection of pyruvate by the formation of the fluorescent pyruvate hydrazone of NBD hydrazine.…”

Section: Melanogaster and C Felis Tps Assay Development For Screementioning

confidence: 99%

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Trehalose‐6‐phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening

Kern¹,

Wolf²,

Bender³

et al. 2012

Insect Molecular Biology

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Trehalose phosphate synthase (EC 2.4.1.15; TPS) is the crucial enzyme for the biosynthesis of trehalose, the main haemolymph sugar of insects, and therefore a potential insecticidal molecular target. In this study, we report the functional heterologous expression of Drosophila melanogaster TPS, the gene identification, full length cDNA cloning and functional expression of cat flea (Ctenocephalides felis) TPS, and the Michaelis–Menten constants for their specific substrates glucose‐6‐phosphate and uridinediphosphate‐glucose. A novel high throughput screening‐compatible TPS assay and its use for the identification of the first potent insect TPS inhibitors from a large synthetic compound collection (>115 000 compounds) is described. One compound class that emerged in this screening, the 4‐substituted 2,6‐diamino‐3,5‐dicyano‐4H‐thiopyrans, was further investigated by analysing preliminary structure–activity relationships. Here, compounds were identified that show low µM to high nM half maximal inhibitory concentrations on insect TPS and that may serve as lead compounds for the development of insecticides with a novel mode of action.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Melanogaster and C Felis Tps Assay Development For Screementioning

confidence: 99%

See 2 more Smart Citations

Trehalose‐6‐phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening

Kern¹,

Wolf²,

Bender³

et al. 2012

Insect Molecular Biology

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Anhydrobiosis in nematodes: Biosynthesis of trehalose

1980

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The pathway for the synthesis of trehalose inAphelenchus auenae was demonstrated. As in other systems, the enzymes involved in the synthesis of trehalose ofA. auenae were found to be trehalose phosphate synthetase (TPS) and trehalose phosphate phosphatase (TPP). Some of the characteristics of trehalose phosphate synthetase were studied. It was found that the rate limiting step in the reaction is catalyzed by TPS. Partially purified TPS was specific for UDPG as the glucosyl donor, and showed a pH optimum of 8.0. The partially purified enzyme has a Km of 0.6 mM and a V,,, of 2.2 x pmoleslmg protein min for glucose-6-phosphate, and a Km of 2.4 mM and V,,, of 2.5 x pmoleslmg protein min for UDPG.

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Anhydrobiosis in nematodes: Control of the synthesis of trehalose during induction

1980

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Control of the synthesis of trehalose during the induction of anhydrobiosis was examined. Kinetic parameters for TPS did not change during the induction, indicating that there was no de novo synthesis of an isozyme. TPS activity doubled in the presence of a mixture of Na+, K+, Mg++, and Ca++ at concentrations normally found in the worms during induction. The concentration of glucose-6-phosphate increased from about 2 mM in wet worms to about 12 mM at the end of induction, and the concentration of UDPG increased from about 1 mM in wet worms to about 9 mM at the end of induction. The concentration of UDPG was found to be rate limiting. Trehalase activity decreased by half during the induction period. The significance of these data as related to the control of the synthesis of trehalose was discussed.

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A coupled spectrophotometric assay for trehalose 6-phosphate synthetase from Dictyostelium discoideum

Cited by 5 publications

References 15 publications

Trehalose‐6‐phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening

Trehalose‐6‐phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening

Anhydrobiosis in nematodes: Biosynthesis of trehalose

Anhydrobiosis in nematodes: Control of the synthesis of trehalose during induction

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