Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine.
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.
Lin
J T
JT
WRRC, ARS, USDA, Albany, California 94710, USA.
McKeon
T A
TA
Goodrich-Tanrikulu
M
M
Stafford
A E
AE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010940:Plant Proteins; D010947:Plants, Toxic; D012278:Ricinus / Q000201:enzymology* / Q000648:ultrastructure
D004791:Enzyme Inhibitors; D010713:Phosphatidylcholines; D010940:Plant Proteins; D015636:Magnesium Chloride; D009243:NAD; D019301:Oleic Acid; D009249:NADP; D000255:Adenosine Triphosphate; D006899:Mixed Function Oxygenases; D002374:Catalase; C094812:phosphatidylcholine 12-monooxygenase