Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver.
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.
Shimazaki
Youji
Y
Department of Physics and Chemistry, Faculti of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Sugawara
Yuki
Y
Manabe
Takashi
T
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D002457:Cell Extracts; D003600:Cytosol / Q000737:chemistry* / Q000201:enzymology* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006597:Fructose-Bisphosphatase / Q000032:analysis / Q000737:chemistry; D007064:L-Iditol 2-Dehydrogenase / Q000032:analysis / Q000737:chemistry; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008291:Malate Dehydrogenase / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D010449:Peptide Mapping; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002457:Cell Extracts; D000804:Angiotensin II; D000324:Adrenocorticotropic Hormone; D007064:L-Iditol 2-Dehydrogenase; D008291:Malate Dehydrogenase; D006597:Fructose-Bisphosphatase