A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase

Ram Rajasekharan, Tapas K. Ray, John E. Cronan

Research output: Contribution to journalArticlepeer-review

Abstract

1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate.

Original languageEnglish (US)
Pages (from-to)376-382
Number of pages7
JournalAnalytical Biochemistry
Volume173
Issue number2
DOIs
StatePublished - Sep 1988

Keywords

  • acylation
  • acyltransferase
  • assay
  • lysophosphatidic acid
  • phosphatidic acid

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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