Biosynthesis of pentosyl lipids by pea membranes

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Abstract
Pea membranes were incubated with UDP-[14C]xylose or UDP-[14C]arabinose and sequentially extracted with chloroform/methanol/water (10:10:3, by volume) and sodium dodecyl sulfate (2% wt/vol). An active epimerase in the membranes rapidly interconverted the 2 pentosyl nucleotides. Chromatographic analysis of the lipid extract revealed that both substrates gave rise to xylose- and arabinose-containing neutral lipids, xylolipid with properties similar to a polyisoprenol monophosphoryl derivative, and highly charged lipid-linked arabinosyl oligosaccharide. When UDP-[14C]pentose or the extracted lipid-linked [14C]arabinosyl oligosaccharide were used as substrates, their 14C was also incorporating into sodium dodecyl sulfate-soluble and -insoluble fractions as major end products. Polyacrylamide-gel electrophoresis of sodium dodecyl sulfate-soluble products indicated the formation of mobile components with MW values between 40,000 and 20,000 (Sepharose CL-6B). The lipid-linked [14C]arabinosyl oligosaccharide possessed properties comparable with those of unsaturated polyisoprenyl pyrophosphoryl derivatives. It was hydrolyzed by dilute acid to a charged product (apparent MW 2300) that could be fractionated in alkali. It was degraded to shorter labeled oligosaccharides by slightly more concentrated acid and eventually to [14C]arabinose as the only labeled component. Susceptibility to acid hydrolysis, and methylation analysis, indicated that the oligosaccharide contained approximately 7 sequential .alpha.-1,5-linked arabinofuranosyl units at the non-reducing end. Several acidic residues appear to be interposed between the terminal arabinosyl units and the charge lipid.