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 luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.
Craig
F F
FF
Amersham International plc, Little Chalfont, Bucks, U.K.
Simmonds
A C
AC
Watmore
D
D
McCapra
F
F
White
M R
MR
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000302:isolation & purification / Q000378:metabolism; D049454:Firefly Luciferin / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D007668:Kidney; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008163:Luminescent Measurements; D013379:Substrate Specificity
D049454:Firefly Luciferin; D008156:Luciferases; D004950:Esterases