Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells
✍ Scribed by Gandelman, Olga ;Allue, Isidro ;Bowers, Keith ;Cobbold, Peter
- Publisher
- John Wiley and Sons
- Year
- 1994
- Weight
- 709 KB
- Volume
- 9
- Category
- Article
- ISSN
- 0884-3996
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✦ Synopsis
In order t o improve calibration o f firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells w e have investigated w h y the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and w h y the decay o f the intracellular signal is remarkably slow. We have shown t h a t inorganic pyrophosphatase greatly depresses the signal in vitro and t h a t micromolar concentrations o f inorganic pyrophosphate, comparable w i t h t h a t in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations o f pyrophosphate depress the signal by inhibiting ATP-binding t o luciferase. Luciferase-injected cells exposed t o extracellular luciferin concentrations above about 100pmol/l (corresponding t o a cytoplasmic level o f c. 5-10pmol/l because o f a transplasmalemmal gradient) show a gradual, irreversible loss o f signal. We attribute this phenomenon (which is not seen in vitro) t o the gradual accumulation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At l o w luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100pmol/l extracellular luciferin, the pyrophosphate level in the cytoplasm fails t o fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders o f magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4pmol/l.