Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non-denaturing two-dimensional gel electrophoresis.
After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.
Shimazaki
Youji
Y
Graduate School of Science and Engineering (Science Section), Venture Business Laboratory, Ehime University, Matsuyama City 790-8577, Japan. yoji@dpc.ehime-u.ac.jp
Miyamoto
Masayuki
M
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D004950:Esterases / Q000737:chemistry*; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification*; D008074:Lipoproteins / Q000737:chemistry* / Q000302:isolation & purification*; D008567:Membranes, Artificial; D011489:Protein Denaturation
D004800:Enzymes, Immobilized; D008055:Lipids; D008074:Lipoproteins; D008567:Membranes, Artificial; D004950:Esterases