Premium
Characterization of oleoyl‐12‐hydroxylase in castor microsomes using the putative substrate, 1‐acyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphocholine
Author(s) -
Lin JiannTsyh,
McKeon Thomas A.,
GoodrichTanrikulu Marta,
Stafford Allan E.
Publication year - 1996
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02523827
Subject(s) - lipidology , phosphocholine , microsome , clinical chemistry , substrate (aquarium) , chemistry , biochemistry , characterization (materials science) , stereochemistry , enzyme , biology , phospholipid , materials science , nanotechnology , membrane , ecology , phosphatidylcholine
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°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 subtrate of oleoyl‐12‐hydroxylase is 2‐oleoyl‐PC.