Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography.
Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.
D002736:Chloroplasts / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D038983:Galactolipids; D005700:Galactosyltransferases / Q000032:analysis; D006017:Glycolipids / Q000378:metabolism*; D008055:Lipids / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet
D038983:Galactolipids; D006017:Glycolipids; D008055:Lipids; D005700:Galactosyltransferases