Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two-dimensional electrophoresis.
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.
Shimazaki
Youji
Y
Department of Physics and Chemistry, Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Sugawara
Yuki
Y
Ohtsuka
Yuki
Y
Manabe
Takashi
T
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004798:Enzymes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D005293:Ferritins / Q000032:analysis; D006597:Fructose-Bisphosphatase / Q000032:analysis / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000032:analysis / Q000737:chemistry; D051379:Mice; D011489:Protein Denaturation; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051137:Selenium-Binding Proteins; D012709:Serum Albumin / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013974:Thyroxine / Q000494:pharmacology; D014168:Transferrin / Q000032:analysis
D002352:Carrier Proteins; D004798:Enzymes; D050556:Fatty Acid-Binding Proteins; D006454:Hemoglobins; D020543:Proteome; D051137:Selenium-Binding Proteins; D012709:Serum Albumin; D014168:Transferrin; D000249:Adenosine Monophosphate; D005293:Ferritins; D008291:Malate Dehydrogenase; D006597:Fructose-Bisphosphatase; D002256:Carbonic Anhydrases; D013974:Thyroxine