Solubilization of arachidonate-CoA ligase from cell membranes, chromatographic separation from nonspecific long-chain fatty acid CoA ligase, and isolation of mutant cell line defective in arachidonate-CoA ligase

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Abstract

The chapter presents a study on solubilization of arachidonate-CoA ligase from cell membranes, chromatographic separation from nonspecific long-chain fatty acid CoA ligase, and isolation of mutant cell line defective in arachidonate-CoA ligase. Two important pieces of evidence suggested the existence of an arachidonate-specific acyl-CoA synthetase, which was different from the previously characterized acyl-CoA synthetase, showing broad specificity for long-chain fatty acids. First, when crude homogenates of platelets were heated to 45 degree for 30 minutes, arachidonoyl-CoA-forming activity was lost four times faster than nonspecific long-chain acyl-CoA synthetase activity. Second, a mutant cell line defective in arachidonate incorporation into phospholipids was isolated and found to lack arachidonoyl, but not nonspecific acyl-CoA synthetase activity. However, the conclusive proof for the existence of an arachidonate specific acyl-CoA synthetase was provided by chromatographic separation of this enzyme activity from the nonspecific long-chain acyl-CoA synthetase. The fatty acid substrate specificity of arachidonoyl-CoA synthetase includes all fatty acids that can subsequently be converted by cyclooxygenase or lipoxygenase to eicosanoids. Nonspecific long-chain acyl-CoA synthetase and arachidonoyl-CoA synthetase activities are assayed.

Original languageEnglish (US)
Pages (from-to)237-242
Number of pages6
JournalMethods in enzymology
Volume187
Issue numberC
DOIs
StatePublished - Jan 1 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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