Simultaneous analysis of esterase and transferase activities in cytosol proteins from the bovine retina by using microscale non-denaturing two-dimensional electrophoresis.
Esterase and transferase activities were analyzed simultaneously after cytosol proteins in the bovine retina were separated by microscale non-denaturing two-dimensional electrophoresis (2-DE). Esterase activity was specifically inhibited by an esterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), and transferase activity was specifically inhibited by a glutathione S-transferase (GST) inhibitor, 2-phenyl-1,2-benziso selenazol-3(2H)-one (ebselen). Both esterase and transferase were precipitated when ammonium sulfate was added to the cytosol up to 50% saturation (50% AS fraction), and were detected in the 50% AS fraction by using the 2-DE. After the cytosol proteins in the 50% AS fraction were separated by using non-denaturing 2-DE, polypeptides of the separated proteins were identified by peptide mass fingerprinting and post-source decay analysis by using MALDI-MS, or by immunoreactivity by using a specific antibody. The spots of esterase and transferase activities in the 2-DE pattern were identified as phosphodiesterase and GST, respectively. This simultaneous analysis of enzyme activities can be applied to screen-specific or non-specific medicines which affect enzyme activities.
Shimazaki
Youji
Y
Department of Chemistry, Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan. yoji@dpc.ehime-u.ac.jp
Hiraka
Yoko
Y
Uesugi
Minatsu
M
Manabe
Takashi
T
D000818:Animals; D001393:Azoles; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004950:Esterases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000032:analysis; D015151:Immunoblotting; D054833:Isoindoles; D016566:Organoselenium Compounds; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D012160:Retina / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013619:Tacrine; D014166:Transferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification
D001393:Azoles; D054833:Isoindoles; D016566:Organoselenium Compounds; C042986:ebselen; D013619:Tacrine; D014166:Transferases; D005982:Glutathione Transferase; D004950:Esterases; D010727:Phosphoric Diester Hydrolases