Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non‐denaturing two‐dimensional gel electrophoresis

Abstract

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.

Determination of Malic Acid Using a Malate Dehydrogenase Reactor After Purification and Immobilization in Non-Denaturing Conditions and Staining with Ponceau S

“…However, it is difficult to recognize protein spots in the non-denaturing 2-DE gel when proteins are detected by reversible staining. Furthermore, we have reported that esterase is immobilized membranes without impairing its activity after extraction, separation, transblotting onto membranes and staining by a dye, direct blue [4]. However, it is difficult to recognize spots possessing enzyme activity on the membranes because the direct blue cannot be removed after staining.…”

Direct analysis of retinal dehydrogenase activity on an electroblotting membrane following separation by non-denaturing two-dimensional electrophoresis

“…So, in order to examine the continuous changes of substrates catalyzed by enzymes, it is preferable that enzymatic reactions are performed on the surface of a support without using chromophores. Since phosphatidylcholine and lipids of high density lipoprotein are hydrolyzed by an esterase on a polyvinylidene difluoride (PVDF) membrane after separation by non-denaturing 2-DE and electroblotting onto the membrane [8], retinoids such as retinylester, retinal, retinol and retinoic acid can be metabolized by enzymes on the membrane. It has been reported that abundant radical molecular ions [M +• ] of retinoids such as retinylester, retinal, retinol, and retinoic acid can be examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) [9,10].…”