A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme.
We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.
Hagishita
T
T
Research Institute for Biological Sciences, Okayama, Kayo-cho, Okayama 716-1241, Japan.
Nishikawa
M
M
Hatanaka
T
T
D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000032:analysis / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
D009596:Nitrophenols; D010743:Phospholipids; D010739:Phospholipase D; C024836:4-nitrophenol