title: Enzyme separation and isozyme heterogeneity analysis using non-denaturing two-dimensional electrophoresis.
abstract: Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6-8) and 1.25% carrier ampholyte (pH 3-10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis.
authors.affiliation: Graduate School of Science and Engineering (Science Section) 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: Kenta
authors.initials: K
authors.lastname: Shimizu
authors.forename: Suzuna
authors.initials: S
authors.lastname: Masaoka
mesh_terms: D043182:Carboxylesterase / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007064:L-Iditol 2-Dehydrogenase / Q000302:isolation & purification; D010088:Oxidoreductases / Q000302:isolation & purification
chemical_list: D004798:Enzymes; D007527:Isoenzymes; D010088:Oxidoreductases; D007064:L-Iditol 2-Dehydrogenase; D043182:Carboxylesterase