Synthesis of Fluorogenic Substrates for Continuous Assay of Phosphatidylinositol-Specific Phospholipase C

To, Zaikova; Av, Rukavishnikov; Gb, Birrell; Griffith, O. Hayes; Jf, Keana

doi:10.1021/bc0001138

Cited by 33 publications

(27 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[10b, 10c, 11a] In contrast, 3-O-methylfluorescein ( MF ; λ max = 472 nm, λ emit = 510 nm, Φ = 0.45) is an enhanced xanthene that can be locked in a non-fluorescent lactone form through a single modification (Scheme 1). Fluorogenic MF derivatives have been used to assay proteases [9b] and phospholipase, [14] but not sulfatases. A few sulfated fluoresceins have been reported as fluorogenic substrates, but were only utilized as a phosphate isostere in phosphatase assays (e.g., to examine phosphatase inhibition).…”

mentioning

confidence: 99%

An Expanded Set of Fluorogenic Sulfatase Activity Probes

2014

View full text Add to dashboard Cite

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3-O-methylfluorescein-sulfate and resorufin-sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species specific sulfatase banding patterns in a gel-resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays, and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.

show abstract

mentioning

confidence: 99%

An Expanded Set of Fluorogenic Sulfatase Activity Probes

2014

View full text Add to dashboard Cite

show abstract

“…Deprotection with trifluoroacetic acid furnished the urea–rhodamine 7 that underwent carbodiimide-mediated coupling with trimethyl lock acid 8 21 to give benzyl-protected 9 . Removal of the benzyl group by catalytic hydrogenation at −5 °C22 afforded acid 10 . Activation of the acid to the succimidyl ester, followed by reaction with alkyl chloride 11 gave the desired probe 1 .…”

Section: Resultsmentioning

confidence: 99%

Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells

et al. 2009

View full text Add to dashboard Cite

Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme–substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse–chase experiments, high-content screening, and numerous other protocols.

show abstract

“…1 H NMR spectra were obtained with NMR spectrometers operating at 300, 360 and 400 MHz. 13 C NMR spectra were obtained at 75 MHz, and 31 P NMR spectra at 146 and 162 MHz. Chemical shifts are referenced directly to tetramethylsilane for 1 H and 13 C NMR, and indirectly to 85 % orthophosphoric acid for 31 P NMR.…”

Section: Determination Of Critical Micelle Concentrationsmentioning

confidence: 99%

“…13 C NMR spectra were obtained at 75 MHz, and 31 P NMR spectra at 146 and 162 MHz. Chemical shifts are referenced directly to tetramethylsilane for 1 H and 13 C NMR, and indirectly to 85 % orthophosphoric acid for 31 P NMR. Mass spectra were recorded on a Micromass (Manchester, UK) Quattro II Triple Quadrupole mass spectrometer.…”

Section: Determination Of Critical Micelle Concentrationsmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorothiolate Analogues of Phosphatidylinositols as Assay Substrates for Phospholipase C

et al. 2007

View full text Add to dashboard Cite

Accurate measurement of phosphatidylinositol-specific phospholipase C (PI-PLC) activity is important in view of the key role of this enzyme in signal-transduction pathways. In this work we synthesized enantiomerically pure phosphorothiolate analogues of all natural PI-PLC substrates, including those of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), 4-phosphate (PI-4-P), 5-phosphate (PI-5-P) and unphosphorylated PI, in both long- and short-chain versions. The enzymatic cleavage of these substrates produces thiol analogues of diacyl glycerol, which can be quantified by UV absorbance after treatment with dipyridyl disulfide. The monodisperse dihexanoyl derivatives are suitable substrates for PI-PLC assay: they give rise to high enzyme activity, and provide excellent linear kinetic responses. For all substrates, we found a good linear correlation between the reaction rate and the amount of enzyme; this indicated the suitability of this assay for enzyme quantification. The short-chain substrates enable the enzyme specificity with variously phosphorylated inositol head groups to be established--unobstructed by substrate aggregation, "scooting" kinetics on micelles, or surface dilution effects. The kinetic results indicated allosteric behavior of PLC for all substrates tested. We found that substrates phosphorylated at the inositol 4-position (phosphorothiolate analogues of PI-4,5-P2 and PI-4-P) displayed very similar kinetic properties, and were cleaved with approximately 20- to 30-fold higher activity than the 4-nonphosphorylated substrates (analogues of PI-5-P and PI). Hence it appears that interactions between the enzyme and the 4-phosphate group of the substrate, but not its 5-phosphate group, is important for PI-PLC catalysis. In addition, the binding affinities of all four substrate types were found to be quite similar; this indicates that the energy of enzyme interaction with the 4-phosphate group is directed almost entirely to catalysis.

show abstract

Synthesis of Fluorogenic Substrates for Continuous Assay of Phosphatidylinositol-Specific Phospholipase C

Cited by 33 publications

References 22 publications

An Expanded Set of Fluorogenic Sulfatase Activity Probes

An Expanded Set of Fluorogenic Sulfatase Activity Probes

Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells

Phosphorothiolate Analogues of Phosphatidylinositols as Assay Substrates for Phospholipase C

Contact Info

Product

Resources

About