Effect of ATP concentration and temperature 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. 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.

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Enhancement of firefly luciferase activity by cytidine nucleotides

Analytical Biochemistry ◽

10.1016/0003-2697(92)90239-4 ◽

1992 ◽

Vol 204 (2) ◽

pp. 283-291 ◽

Cited By ~ 25

Author(s):

Sharon R. Ford ◽

Marliese S. Hall ◽

Franklin R. Leach

Keyword(s):

Luciferase Activity ◽

Firefly Luciferase ◽

Firefly Luciferase Activity

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One-step measurement of firefly luciferase activity in yeast

Yeast ◽

10.1002/yea.1024 ◽

2003 ◽

Vol 20 (13) ◽

pp. 1109-1113 ◽

Cited By ~ 54

Author(s):

P. Leskinen ◽

M. Virta ◽

M. Karp

Keyword(s):

Luciferase Activity ◽

Firefly Luciferase ◽

One Step ◽

Firefly Luciferase Activity

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Berberine inhibits adipocyte differentiation, proliferation and adiposity through down-regulating galectin-3

Scientific Reports ◽

10.1038/s41598-019-50103-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Can Wang ◽

Yan Wang ◽

Shu-Rong Ma ◽

Zeng-Yan Zuo ◽

Yan-Bin Wu ◽

...

Keyword(s):

High Fat Diet ◽

Adipocyte Differentiation ◽

Luciferase Activity ◽

Firefly Luciferase ◽

Preadipocyte Differentiation ◽

Galectin 3 ◽

Epididymal White Adipose Tissue ◽

Treatment Of Obesity ◽

Differentiation And Proliferation ◽

Firefly Luciferase Activity

Abstract This study is designed to investigate the effects of berberine (BBR) on galectin-3 (Gal-3) and the relationships to its suppressive activities on adipocyte differentiation, proliferation and adiposity. Our results showed that BBR greatly suppressed the differentiation and proliferation of mouse primary preadipocytes isolated from epididymal white adipose tissue (eWAT), during which the expression level of Gal-3 was down-regulated significantly. Overexpression of Gal-3 totally abolished the suppressive activities of BBR on Gal-3 expression, preadipocyte differentiation and proliferation. BBR reduced Gal-3 promoter activity, destabilized its mRNA and inhibited firefly luciferase activity of a recombinant plasmid containing the Gal-3 3′ untranslated region (UTR). Furthermore, BBR up-regulated microRNA (miRNA) let-7d expression and the suppressive activity on Gal-3 3′UTR was abolished by point mutation on the let-7d binding site. In mice fed a high-fat diet (HFD), BBR up-regulated let-7d and down-regulated Gal-3 expression in eWAT; it also suppressed adipocyte differentiation and proliferation and reduced adiposity greatly. In summary, our study proves that BBR inhibits the differentiation and proliferation of adipocytes through down-regulating Gal-3, which is closely associated with its anti-obesity effect. Our results may support the future clinical application of BBR for the treatment of obesity or related diseases.

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