title: 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.
abstract: 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.
authors.affiliation: Department of Chemistry, Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
authors.forename: Youji
authors.initials: Y
authors.lastname: Shimazaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Manabe
mesh_terms: 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
chemical_list: D003902:Detergents; D000444:Aldehyde Dehydrogenase; D043182:Carboxylesterase