Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography.
An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.
Iwasaki
Yugo
Y
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8610, Japan. iwasaki@agr.nagoya-u.ac.jp
Masayama
Atsushi
A
Mori
Akihiro
A
Ikeda
Chika
C
Nakano
Hideo
H
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D048448:Phenylcarbamates / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D048448:Phenylcarbamates; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D007294:Inositol; D010739:Phospholipase D