A facile purification procedure of phospholipase D from cabbage and its characterization
| Autor: | Renate Ulbrich-Hofmann, Romy Lambrecht |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Chromatography
Molecular mass Elution Substrate (chemistry) chemistry.chemical_element Brassica Calcium Chromatography Ion Exchange Biochemistry Micelle Molecular Weight Electrophoresis Kinetics Isoelectric point chemistry Critical micelle concentration Chromatography Gel Phospholipase D lipids (amino acids peptides and proteins) Electrophoresis Polyacrylamide Gel |
| Zdroj: | Biological chemistry Hoppe-Seyler. 373(2) |
| ISSN: | 0177-3593 |
| Popis: | Phospholipase D (PLD), an enzyme predestined for the preparation of new phospholipids, was isolated from cabbage and purified in a highly efficient way by using a combination of hydrophobic chromatography and a specific calcium effect. In the presence of calcium ions (50mM), PLD is bound from the crude enzyme solution to Octyl-Sepharose and subsequently selectively eluted by removing the calcium ions. The obtained enzyme is electrophoretically pure (95%), its molecular mass and isoelectric point were determined to be 87,000 Da and 4.7, respectively. The purified enzyme was kinetically characterized by use of mixed phosphatidylcholine-SDS micelles as well as the short-chain lecithins 1,2-dihexanoyl- and 1,2-diheptanoyl-sn-glycero-3-phosphocholine as substrates. A hyperbolic upsilon/[S]-characteristic was obtained for the mixed micellar system, whereas the upsilon/[S] curves of the short-chain lecithins reflect the dependence of velocity on the physical state of the substrate. A small velocity increase was observed up to a critical substrate concentration near the critical micelle concentration, from where the velocity increases hyperbolically. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|