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<center>luciferyl adenylate + [[Oxygen|O<sub>2</sub>]] → oxyluciferin + [[Adenosine monophosphate|AMP]] + light</center>
<center>luciferyl adenylate + [[Oxygen|O<sub>2</sub>]] → oxyluciferin + [[Adenosine monophosphate|AMP]] + light</center>


The reaction is very energy efficient: nearly all of the energy input into the reaction is transformed into light. As a comparison, the [[light bulb|incandescent light bulb]] loses about 90% of its [[energy]] to [[heat]].<ref>General Electric TP-110, page 23, table.</ref>
The reaction is very energy efficient: nearly all of the energy input into the reaction is transformed into light. As a comparison, the [[light bulb|incandescent light bulb]] loses about 90% of its [[energy]] to [[heat]]<ref>General Electric TP-110, page 23, table.</ref>.


'''Luciferin''' and '''luciferase''' are not specific molecules. They are generic terms for a substrate and its associated [[enzyme]] (or [[protein]]) that [[catalyze]] a light-producing reaction. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the [[firefly|fireflies]], although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom ''([[Omphalotus olearius]])'' and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the [[abdominal]] [[trachea]]. The luciferases of fireflies - of which there are over 2000 [[species]] - and of the [[Elateroidea]] (fireflies, click beetles and relatives) in general - are diverse enough to be useful in [[molecular phylogeny]]. The most thoroughly studied luciferase is that of the [[Photinini]] firefly ''Photinus pyralis'', which has an optimum pH of 7.8 <ref>Steghens J.-P., Min K.-L., Bernengo J.-C. (1998) Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra; ''Biochem. J.'' '''336''', 109-113; http://www.biochemj.org/bj/336/0109/3360109.pdf </ref>.
'''Luciferin''' and '''luciferase''' are not specific molecules. They are generic terms for a substrate and its associated [[enzyme]] (or [[protein]]) that [[catalyze]] a light-producing reaction. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the [[firefly|fireflies]], although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom ''([[Omphalotus olearius]])'' and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the [[abdominal]] [[trachea]]. The luciferases of fireflies - of which there are over 2000 [[species]] - and of the [[Elateroidea]] (fireflies, click beetles and relatives) in general - are diverse enough to be useful in [[molecular phylogeny]]. The most thoroughly studied luciferase is that of the [[Photinini]] firefly ''Photinus pyralis'', which has an optimum pH of 7.8 <ref>Steghens J.-P., Min K.-L., Bernengo J.-C. (1998) Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra; ''Biochem. J.'' '''336''', 109-113; http://www.biochemj.org/bj/336/0109/3360109.pdf </ref>.
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<ref name=introanimation>Promega Corporation. [http://www.promega.com/multimedia/bioLum01.htm Introduction to Bioluminescent Assays]. (Animation, requires Flash player)</ref>.
<ref name=introanimation>Promega Corporation. [http://www.promega.com/multimedia/bioLum01.htm Introduction to Bioluminescent Assays]. (Animation, requires Flash player)</ref>.


In biological research, luciferase commonly is used as a reporter to assess the [[transcription|transcriptional]] activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a [[promoter]] of interest<ref name=fanwood> [http://dx.doi.org/10.1089/adt.2006.053 Fan, F. and Wood, K. (2007) Bioluminescent Assays for High-Throughput Screening. ''ASSAY and Drug Development Technologies'' '''5''', 127–136.] </ref>. Luciferase can also be used detect the level of cellular [[Adenosine triphosphate|ATP]] in cell viability assays or for kinase activity assays<ref name=fanwood/><ref>[http://www.promega.com/pnotes/100/16620_22/16620_22.pdf Meisenheimer, P.L. ''et al''. (2008) Luminogenic enzyme substrates: The basis for a new paradigm in assay design. ''Promega Notes'' '''100''', 22–26.]</ref>. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect [[caspase |caspase]] activity and [[cytochrome P450|cytochrome P450]] activity, among others.<ref name=introanimation/><ref name=fanwood/>
In biological research, luciferase commonly is used as a reporter to assess the [[transcription|transcriptional]] activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a [[promoter]] of interest<ref name=fanwood> [http://dx.doi.org/10.1089/adt.2006.053 Fan, F. and Wood, K. (2007) Bioluminescent Assays for High-Throughput Screening. ''ASSAY and Drug Development Technologies'' '''5''', 127–136.] </ref>. Luciferase can also be used detect the level of cellular [[Adenosine triphosphate|ATP]] in cell viability assays or for kinase activity assays<ref name=fanwood/><ref>[http://www.promega.com/pnotes/100/16620_22/16620_22.pdf Meisenheimer, P.L. ''et al''. (2008) Luminogenic enzyme substrates: The basis for a new paradigm in assay design. ''Promega Notes'' '''100''', 22–26.]</ref>. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect [[caspase |caspase]] activity and [[cytochrome P450|cytochrome P450]] activity, among others<ref name=introanimation/><ref name=fanwood/>.


Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive [[CCD camera]].This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models.<ref>[http://cancerres.aacrjournals.org/cgi/content/abstract/63/21/7042?ijkey=86d5632b7fba763375a6782a1cd2e7e40bedd784 Lyons, S.K. ''et al''. (2003)The generation of a conditional reporter that enables bioluminescence imaging of Cre/loxP-Dependent tumorigenesis in mice. ''Cancer Res''.'''63''', 7042–7046.]</ref><ref>[http://dx.doi.org/10.1158/0008-5472.CAN-05-3827 Becher, O.J. and Holland, E.C. (2006) Genetically engineered models have advantages over xenografts in preclinical studies. ''Cancer Res''. '''66''', 3355–3359.]</ref>
Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive [[CCD camera]].This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models<ref>[http://cancerres.aacrjournals.org/cgi/content/abstract/63/21/7042?ijkey=86d5632b7fba763375a6782a1cd2e7e40bedd784 Lyons, S.K. ''et al''. (2003)The generation of a conditional reporter that enables bioluminescence imaging of Cre/loxP-Dependent tumorigenesis in mice. ''Cancer Res''.'''63''', 7042–7046.]</ref><ref>[http://dx.doi.org/10.1158/0008-5472.CAN-05-3827 Becher, O.J. and Holland, E.C. (2006) Genetically engineered models have advantages over xenografts in preclinical studies. ''Cancer Res''. '''66''', 3355–3359.]</ref>.
Luciferase can be used in [[blood bank]]s to determine if [[red blood cell]]s are starting to break down. [[Forensic]] investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on [[protein denaturation]], testing the protective capacities of [[heat shock proteins]]. The opportunities for using luciferase continue to expand. {{Fact|date=September 2008}}
Luciferase can be used in [[blood bank]]s to determine if [[red blood cell]]s are starting to break down. [[Forensic]] investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on [[protein denaturation]], testing the protective capacities of [[heat shock proteins]]. The opportunities for using luciferase continue to expand. {{Fact|date=September 2008}}

Revision as of 19:55, 2 October 2008

Firefly luciferase
Crystal structure of Photinus pyralis firefly luciferase
Identifiers
SymbolFirefly luciferase
PDB1LCI
UniProtP08659
Other data
EC number1.13.12.7
Search for
StructuresSwiss-model
DomainsInterPro

Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. The most famous one is firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis. In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment), sometimes involving adenosine triphosphate (ATP). The rates of this reaction between luciferin and oxygen are extremely slow until they are catalyzed by luciferase, often mediated by the presence of calcium ions (an analog of muscle contraction).[1] The reaction takes place in two steps:

luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light

The reaction is very energy efficient: nearly all of the energy input into the reaction is transformed into light. As a comparison, the incandescent light bulb loses about 90% of its energy to heat[2].

Luciferin and luciferase are not specific molecules. They are generic terms for a substrate and its associated enzyme (or protein) that catalyze a light-producing reaction. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the fireflies, although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. The luciferases of fireflies - of which there are over 2000 species - and of the Elateroidea (fireflies, click beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny. The most thoroughly studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8 [3].

Applications

Luciferase can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Mice, silkworms, and potatoes are just a few organisms that have already been engineered to produce the protein. [citation needed]

In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes [4].

In biological research, luciferase commonly is used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest[5]. Luciferase can also be used detect the level of cellular ATP in cell viability assays or for kinase activity assays[5][6]. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase activity and cytochrome P450 activity, among others[4][5].

Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive CCD camera.This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models[7][8].

Luciferase can be used in blood banks to determine if red blood cells are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand. [citation needed]

See also

References

  1. ^ IUBMB Enzyme Nomenclature Renilla luciferin reaction is triggered by calcium ions. (cited September 6, 2006)
  2. ^ General Electric TP-110, page 23, table.
  3. ^ Steghens J.-P., Min K.-L., Bernengo J.-C. (1998) Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra; Biochem. J. 336, 109-113; http://www.biochemj.org/bj/336/0109/3360109.pdf
  4. ^ a b Promega Corporation. Introduction to Bioluminescent Assays. (Animation, requires Flash player)
  5. ^ a b c Fan, F. and Wood, K. (2007) Bioluminescent Assays for High-Throughput Screening. ASSAY and Drug Development Technologies 5, 127–136.
  6. ^ Meisenheimer, P.L. et al. (2008) Luminogenic enzyme substrates: The basis for a new paradigm in assay design. Promega Notes 100, 22–26.
  7. ^ Lyons, S.K. et al. (2003)The generation of a conditional reporter that enables bioluminescence imaging of Cre/loxP-Dependent tumorigenesis in mice. Cancer Res.63, 7042–7046.
  8. ^ Becher, O.J. and Holland, E.C. (2006) Genetically engineered models have advantages over xenografts in preclinical studies. Cancer Res. 66, 3355–3359.


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