Analysis of enzyme activity regulation by non-denaturing electrophoresis and application of this regulation for enzyme reactor production.
Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities.
Department of Chemistry and Biology, Graduate School of Science and Engineering and Venture Business Laboratory, Ehime University , Matsuyama , Japan.
Youji
Y
Shimazaki
Shizuka
S
Miki
D000818:Animals; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004975:Ethacridine / Q000737:chemistry / Q000494:pharmacology; D007525:Isoelectric Focusing; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D063347:Native Polyacrylamide Gel Electrophoresis; D010072:Oxamic Acid / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007770:L-Lactate Dehydrogenase; D004950:Esterases; D010072:Oxamic Acid; D004975:Ethacridine