Activity of Phospholipase C in Two-Phase Systems
| Autor: | Regine Haftendorn, Renate Ulbrich-Hofmann |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Acylation
Biophysics Buffers Sensitivity and Specificity Biochemistry Micelle Diglycerides chemistry.chemical_compound Hydrolysis Bacillus cereus Phosphatidylcholine Molecular Biology Micelles Chloroform Aqueous solution Chromatography Phospholipase C Chemistry Cell Biology Solutions Kinetics Type C Phospholipases Reagent Phosphatidylcholines Emulsions lipids (amino acids peptides and proteins) Chromatography Thin Layer Deoxycholic Acid |
| Zdroj: | Analytical Biochemistry. 306:144-147 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2002.5692 |
| Popis: | Although phospholipase C (PLC) is known to be activated by water-insoluble organic solvents, most activity assays have been designed to work in an aqueous milieu. Here a sensitive method is described for the determination of PLC activity in two-phase systems. The assay is based on the hydrolysis of phosphatidylcholine (PC) in chloroform/buffer. The initial rates of the reaction are determined by densitometric quantification of the product 1,2-diacylglycerol after its separation by high-performance TLC and staining with a CuSO4/H3PO4 or p-methoxybenzaldehyde/H2SO4 reagent. The method is examined for the determination of Vmax and Km values of PCs with varying length acyl chains (C10-C18). The comparison of the kinetic parameters with the Vmax and Km values of the same substrates in the conventional titrimetric assay, using sodium deoxycholate for micellization of PC, demonstrates the high efficiency of PLC in the two-phase emulsion system. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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