Regulatory effects of ATP and luciferin on firefly luciferase activity

Abstract

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. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: Assessment of an oral agent that stimulates erythropoietin production

“…injection with estimated tissue levels of 60-180 M, with somewhat lower levels in the brain, at the doses used here (26,27). These levels exceed the firefly luciferase luciferin K m (22), and luciferase activity has been successfully imaged in a wide variety of tissues (including brain) by using protocols similar to the one used by us (10,28,29). Nonetheless, the intracellular concentrations of luciferin achieved in different tissues after systemic administration are not known, and differences in luciferin availability might confound luciferase-based imaging in certain settings.…”

“…Therefore, changes in these three factors could potentially complicate the interpretation of bioluminescent images obtained with the ROSA26 ODD-Luc͞ϩ mice. Fortunately, the firefly luciferase K m for oxygen and ATP are Ϸ1-10 M and Ϸ50 M respectively (22,23,and A. Campbell,personal communication). In contrast, the oxygen K m for the HIF prolyl hydroxylases are Ϸ200 M, and cellular ATP levels are estimated to be Ϸ1 mM (24).…”

Glycerol 3-phosphate-induced ATP production in intact mitochondria from pancreatic B-cells

“…Since the ATP produced after 10 min of incubation is around 0.1 nmol, as judged from internal ATP standards, the sensitivity may be increased further by increasing the concentration of luciferase or luciferin. Care must be taken, however, to keep the linear dose-response relation of the signal [20]. Provided that proper measures are taken the amount of mitochondria per sample may be substantially reduced.…”

Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase

“…2A). The transient peak above the plateau just after the addition of luciferin is likely to re£ect the transition to a new equilibrium binding state of luciferase with its two substrates [19]. There was a linear relationship between ATP perfused into permeabilized cells and photon emission from 1 to 10 mM ATP (Fig.…”