A Compilation of Protocols to Visualize Enzymes Following Electrophoretic Separation

Rothe, Gunter M.

doi:10.1007/978-3-642-79069-0_6

Electrophoresis of Enzymes 1994

DOI: 10.1007/978-3-642-79069-0_6

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A Compilation of Protocols to Visualize Enzymes Following Electrophoretic Separation

Gunter M. Rothe

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Cited by 4 publications

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Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two‐dimensional electrophoresis

et al. 2003

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Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.

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Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two‐dimensional electrophoresis

et al. 2003

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Nondenaturing two‐dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

2004

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After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.

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Activity and sequence structure analysis of cytosolic dehydrogenase by mass spectrometry after separation by nondenaturing two-dimensional electrophoresis

Shimazaki

Sugawara

2004

Analytical Biochemistry

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A Compilation of Protocols to Visualize Enzymes Following Electrophoretic Separation

Cited by 4 publications

References 106 publications

Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two‐dimensional electrophoresis

Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two‐dimensional electrophoresis

Nondenaturing two‐dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

Activity and sequence structure analysis of cytosolic dehydrogenase by mass spectrometry after separation by nondenaturing two-dimensional electrophoresis

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