酵素電気泳動像、ザイモグラム

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2013/12/06 03:59:44」(JST)

wiki en

Zymography is a electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. Three types of zymography are used; in gel zymography, in situ zymography and in vivo zymography^[1]

General Procedure[edit]

Gel zymography[edit]

Samples are prepared in a standard, non-reducing loading buffer for SDS-PAGE. No reducing agent or boiling are necessary since these would interfere with refolding of the enzyme. A suitable substrate (commonly gelatin or casein) is embedded in the resolving gel during preparation of the acrylamide gel. Following electrophoresis, the SDS is removed from the gel (or zymogram) by incubation in unbuffered Triton X-100, followed by incubation in an appropriate digestion buffer, for an optimized length of time at 37°C. The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme.

Variations on the standard protocol[edit]

The standard protocol may require modifications depending on the sample enzyme; for instance, D. melanogaster digestive glycosidases generally survive reducing conditions (i.e. the presence of 2-mercaptoethanol or DTT), and to an extent, heating. Indeed, the separations following heating to 50°C tend to exhibit a substantial increase in band resolution, without appreciable loss of activity.^[2]^[3]

A common protocol used in the past for zymography of α-amylase activity was the so-called starch film protocol of W.W. Doane. Here a native PAGE gel was run to separate the proteins in a homogenate. Subsequently, a thin gel with starch dissolved (or more properly, suspended) in it was overlaid for a period of time on top of the original gel.^[4] The starch was then stained with Lugol's iodine.

Gel zymography is often used for the detection en analysis of enzymes produced by microorganisms.^[5] This has led to variations on the standard protocol e.g. mixed-substrate zymography.^[1]

Plasmodium knowlesi hemoglobinase imprint.^[6]

Reverse zymography copolymerizes both the substrate and the enzyme with the acrylamide, and is useful for the demonstration of enzyme inhibitor activity. Following staining, areas of inhibition are visualized as dark bands against a clear (or lightly stained) background.

In imprint technique, the enzyme is separated by native gel electrophoresis and the gel is laid on top of a substrate treated agarose.^[6]

Zymography can also be applied to other types of enzymes, including xylanases, lipases and chitinases.

References[edit]

^ ^a ^b Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013). "Zymography methods for visualizing hydrolytic enzymes." Nat Methods. 10:211-220. doi:10.1038/nmeth.2371. PMID 23443633
^ Martínez TF, Alarcón FJ, Díaz-López M, Moyano FJ (2000). "Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch.". Electrophoresis 21 (14): 2940–2943. doi:10.1002/1522-2683(20000801)21:14<2940::AID-ELPS2940>3.0.CO;2-S. PMID 11001307.
^ Snoek-van Beurden PA, Von den Hoff JW (2005). "Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors". Biotechniques 38 (1): 73–83. doi:10.2144/05381RV01. PMID 15679089.
^ Doane WW (1969). "Amylase variants in Drosophila melanogaster: linkage studies and characterization of enzyme extracts". J Exp Zool 171 (3): 31–41. PMID 5348624.
^ Lantz MS, Ciborowski P (1994). "Zymographic techniques for detection and characterization of microbial proteases.". Methods Enzymol. 235: 563–594. doi:10.1016/0076-6879(94)35171-6. PMID 8057927.
^ ^a ^b Hempelmann, E., Putfarken, B., Rangachari, K., Wilson, R.J.M. (1986). "Immunoprecipitation of malarial acid endopeptidase". Parasitology 92 (02): 305–312. doi:10.1017/S0031182000064076. PMID 3520446.

English Journal

Functional characterization of fibrinolytic metalloproteinases (colombienases) isolated from Bothrops colombiensis venom.

Girón ME, Guerrero B, Salazar AM, Sánchez EE, Alvarez M, Rodríguez-Acosta A.SourceLaboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas, Venezuela.
Toxicon : official journal of the International Society on Toxinology.Toxicon.2013 Nov;74:116-26. doi: 10.1016/j.toxicon.2013.07.025. Epub 2013 Aug 17.
Researchers trying to improve the safety and efficacy of fibrinolytic therapy have been isolating fibrinolytic enzymes from snake venoms. Two fibrinolytic enzymes, colombienases 1 and 2, with significant activity have been isolated from the venom of Bothrops colombiensis.METHODS: The colombienases w
PMID 23958522

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2.

Oberoi HS, Rawat R, Chadha BS.SourceCentral Institute of Post-Harvest Engineering and Technology, P.O. PAU, Ludhiana, 141 004, India, hari_manu@yahoo.com.
Antonie van Leeuwenhoek.Antonie Van Leeuwenhoek.2013 Oct 25. [Epub ahead of print]
Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-
PMID 24158534

Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression.

Ushasree MV, Vidya J, Pandey A.SourceBiotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, 695 019, Kerala, India, ushusbt@gmail.com.
Biotechnology letters.Biotechnol Lett.2013 Sep 28. [Epub ahead of print]
A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. c
PMID 24078121