Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine

Lipids. 1996 Jun;31(6):571-7. doi: 10.1007/BF02523827.

Abstract

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5 degrees C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual substrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Catalase / pharmacology
  • Chromatography, High Pressure Liquid
  • Enzyme Inhibitors / pharmacology
  • Gas Chromatography-Mass Spectrometry
  • Hydrogen-Ion Concentration
  • Magnesium Chloride / pharmacology
  • Microsomes / enzymology*
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / metabolism*
  • NAD / metabolism
  • NADP / metabolism
  • Oleic Acid / metabolism
  • Phosphatidylcholines / metabolism*
  • Plant Proteins
  • Plants, Toxic*
  • Ricinus / enzymology*
  • Ricinus / ultrastructure

Substances

  • Enzyme Inhibitors
  • Phosphatidylcholines
  • Plant Proteins
  • Magnesium Chloride
  • NAD
  • Oleic Acid
  • NADP
  • Adenosine Triphosphate
  • Mixed Function Oxygenases
  • Catalase
  • phosphatidylcholine 12-monooxygenase