Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.
To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.
Shimazaki
Youji
Y
Department of Chemistry, Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Manabe
Takashi
T
D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D043182:Carboxylesterase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D008099:Liver / Q000201:enzymology*; D051379:Mice; D021241:Spectrometry, Mass, Electrospray Ionization
D003902:Detergents; D000444:Aldehyde Dehydrogenase; D043182:Carboxylesterase