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research plasmid DNA plasmid DNA plasmid DNA plasmid DNA plasmid DNA plasmid DNA adenovirus adenovirus
adenovirus adenovirus adenovirus adenovirus adenovirus lactate lactate lactate lactate lactate lactate lactate lactate
chemiluminescent chemiluminescent chemiluminescent chemiluminescent chemiluminescent TMB TMB TMB
chemiluminescent TMB TMB TMB TMB TMB TMB TMB genomic genomic genomic genomic genomic genomic RNA
RNA RNA RNA RNA RNA RNA RNA western blotting western blotting western blotting western blotting protein assay
protein assay protein assay protein assay protein assay SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE
luciferase luciferase luciferase luciferase luciferase luciferase luciferase MTT MTT MTT MTT MTT MTT MTT LDH
LDH LDH LDH LDH LDH LDH cell injury cell injury cell injury cell injury cell injury cell proliferation cell proliferation
galactosidase galactosidase galactosidase galactosidase galactosidase galactosidase competent cell competent cell
competent cell competent cell biomedical research service biomedical research service biomedical research
Exploration of luciferase-based light reactions

Firefly luciferase from the firefly Photinus pyralis is an interesting oxidative enzyme used in bioluminescence. Luciferase (LUC) encoded by the firefly luciferase
gene is widely used as a sensitive reporter enzyme for the study of transcriptional regulation in molecular biology and tissue imaging in translational research. In
the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Light emission peaks in several seconds at 560 nm when the
reaction is conducted at pH 7.8.

The light reaction takes place in two steps: (1) luciferin + ATP → luciferyl adenylate + PPi; (2) luciferyl adenylate + O2 → oxyluciferin + AMP + light. During this
reaction cascade, luciferase undergoes a conformational change, ensuring that water is excluded from the reaction and does not hydrolyze ATP. Recent research
suggests that the interactions between the excited state product and nearby residues can force the oxyluciferin into a higher energy form, causing the emission of
a yellow-green light. Interestingly, luciferase can replace fatty acyl-CoA synthetase in converting long-chain fatty acids to fatty-acyl CoA, which is the entering
substrate for fatty acid beta oxidation. This is because both luciferase and fatty acyl-CoA synthetase share a similar catalytic mechanism in converting ATP to AMP.

Since the enzyme catalyzes, in the presence of ATP, the oxidation of luciferin with concomitant emission of light, which can be conveniently measured by at
luminometer or scintillation counter, this molecular mechanism can be applied for convenient and sensitive
ATP/ADP/AMP measurement. In addition, the principle
can be coupled to substrate-level phosphorylation for enzyme activity assay. For instance, pyruvate kinase converts phosphoenolpyruvate to pyruvate with
concomitant production of one ATP. Luciferase can thus be coupled to the pyruvate kinase reaction for determination of
pyruvate kinase activity. Similarly, the
reaction can also be utilized to determine the activity of
Adenylate kinase (ADK) and Nucleoside diphosphate kinase (NDK).

A more recent application of the luciferase-based technology is in the area of whole animal imaging or in vivo imaging, which is a powerful technique for cell
tracking. Different types of cells have been genetically engineered to express a luciferase. Implantation of these luciferase-marked cells then allows non-invasive
imaging inside a live animal using a sensitive charge-coupled device camera. A major drawback is that environmental factors may interfere with bioluminescence
intensity, and thus the intensity of the obtained signal may be strongly influenced by tissue transparency and permeability, blood flow, and the pH of body fluids.
Future biomedical engineering may overcome this technical limitation.
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