Lipids

, Volume 32, Issue 7, pp 783–788 | Cite as

Chromogenic assay for phospholipase D from Streptomyces chromofuscus: Application to the evaluation of substrate analogs

  • Paul J. Hergenrother
  • Michelle K. Haas
  • Stephen F. Martin
Article

Abstract

A rapid and convenient chromogenic assay for phospholipase D from Streptomyces chromofuscus (PLDSc) has been developed that converts the choline generated from the enzyme-catalyzed hydrolysis of phospholipids into a chromogenic dye. By quenching the reaction with EDTA at defined times, an initial rate curve is produced from which a kcat and Km can be readily derived. This assay has been applied to the biological evaluation of several substrate analogs, all of which appear to be activators rather than substrates or inhibitors of this enzyme. Performing the assay in 96-well microtiter plates allows for the easy screening of potential effectors of this enzyme.

Abbreviation

CMC

critical micelle concentration

DAG

diacylglycerol

PA

phosphatidic acid

PAN

1-(2-pyridyl(azo)-2-naphthol

PC

phosphatidylcholine

PLD

phospholipase D

PLDSc

phospholipase D from Streptomyces chromofuscus

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Copyright information

© AOCS Press 1997

Authors and Affiliations

  • Paul J. Hergenrother
    • 1
  • Michelle K. Haas
    • 1
  • Stephen F. Martin
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of Texas at AustinAustin

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