Application of capillary electrophoresis to determination of enzyme activity and other properties of phosphatidylinositol-specific phospholipase C.
This paper describes a method for monitoring enzymatic activity using micellar electrokinetic chromatography (MEKC). MEKC is used to analyze the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) with convenience and precision. PI specific PLC enzyme converts phosphatidylinositol (PI) to diacylglycerol (DAG) and inositol 1,2-cyclic phosphate (IP). The assay system developed is based on monitoring both the breakdown of substrate and the formation of products simultaneously. To obtain the best separation for the substrate PI and product DAG, we investigated changes in concentration of electrolyte buffer and SDS as well as in pH of the electrolyte buffer. Since the structures of the substrate and products are different, the reaction could be monitored easily by MEKC mostly based on hydrophobicity. Under the separation conditions developed, we investigated the enzymatic activity of PI-PLC depending on the concentrations of phosphatidylinositol, various divalent cations and the reaction temperature. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to confirm the molecular masses of the substrate PI and products DAG and IP. The results of this study show that capillary electrophoresis can be widely applied to analyze and characterize many other enzymes since capillary electrophoresis has several advantages as follows, simplicity, speed, no need for radiolabelled substrate, small sample volumes and sufficient accuracy.
Suh
M J
MJ
Doping Control Center, Korea Institute of Science and Technology, Seoul, South Korea.
Kim
Y S
YS
Yoo
Y S
YS
D002413:Cations, Divalent / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008823:Micelles; D008970:Molecular Weight; D009994:Osmolar Concentration; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D010738:Type C Phospholipases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D002413:Cations, Divalent; D008823:Micelles; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase