Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

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Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells.

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak lumi...

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Regulatory effects of ATP and luciferin on firefly luciferase activity.

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. Hig...

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Firefly luciferase can use L-luciferin to produce light.

L-Luciferin is a competitive inhibitor of firefly luciferase with a K1 between 3 and 4 microM. Furthermore L-luciferin can serve as an alternative substrate for light production. Catalysis of L-luciferin can be observed in the absence of, or at low concentrations of, D-luciferin. The light production from L-luciferin increases slowly (maximal half-time 8 min) to a stable plateau. At low concent...

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Assay of rat plasma pyruvate kinase activity with luciferin-luciferase.

An assay method for pyruvate kinase in rat plasma is described. Plasma samples were incubated with ADP and phosphoenolpyruvate in Tris buffer solution. The ATP produced by pyruvate kinase was measured by photocounting after the addition of a commercially available luciferin-luciferase preparation. Interference by ATP or adenylate kinase originally present in the sample was removed by a high deg...

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Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase

Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain...

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ژورنال

عنوان ژورنال: Biochemical Journal

سال: 1991

ISSN: 0264-6021,1470-8728

DOI: 10.1042/bj2760637