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A method for a large scale extraction of phosphoglycosphingolipids from the leaves of Nicotiana tabacum L. has been developed. The phosphosphingolipid concentrate consists of a dozen or more polar lipids as judged by thin layer chromatography. Two of these lipids were purified by chromatography on porous silica beads and partially characterized. These lipids are formulated as: N-acetylglucosamidoglucuronidoinositol phosphorylceramide and glucosamido,glucuronidoinositol phosphorylceramide. Although not fully characterized, the other lipids in the concentrate are inositol-containing phosphosphingolipids with a higher carbohydrate content.Glycosphingolipids are thought to play a significant role in cell surface function in animals. Considerable information is available concerning their chemistry, biosynthesis, and degradation (38). The neutral glycosphingolipids are present in most animal cells and are composed of uncharged heterooligosaccharides of various chain lengths glycosidically linked to ceramide (N-fattyacylsphingosine). Ceramide-monohexosides (cerebrosides) have been characterized from higher plants, animals and fungi (38). Neutral ceramide-oligosaccharides, to our knowledge, have not been reported in higher plants, although a ceramide-tetrasaccharide has been isolated from the fungus, Neurospora crassa (19). Gangliosides, negatively charged glycosphingolipids, which derive their charge from one or more sialic groups glycosidically linked to the oligosaccharide portions of various ceramide-oligosaccharides (38), are widespread in animal cells but have not been reported to occur in plants.A negatively charged glycosphingolipid that does occur in plants is phytoglycolipid, so named by Carter and co-workers (6) who have carried out pioneering investigations in this area. They showed that phytoglycolipid occurred in seeds from corn, flax, and soybeans (6), as well as in bean leaves (11). Phytoglycolipid is apparently unique to plants, although related substances have been reported in fungi (19,29 . The leaves were packed in crushed ice immediately after harvesting and were kept at 0 to 5 C through the homogenization step which was carried out within 3 to 6 hours after harvest. To the eluate one-half volume of CH3OH was added, and the resulting precipitate was allowed to sediment for 2 days at 5 C. The supernatant was removed by siphoning and was kept at -20 C for 2 more days to obtain more precipitate. The combined 5 C and -20 C sedimented precipitate slurry was centrifuged to remove the supernatant remaining after siphoning. The precipitate obtained was air dried and stored in a vacuum dessicator at 5 C (stage V). Plant Physiol. Vol. 55, 1975 Preparation of Na-phosphosphingolipid Concentrate. In a typical experiment, stage V precipitate (1.06 mmole of P) was suspended in 100 ml of pyridine-H20 (3:7, v/v) and passed through a 40-ml column of Chelex 100 resin (Nat, 100-200 mesh, Bio-Rad Laboratories) equilibrated with the same solvent adjusted to pH 8.8. The column was further eluted with 70 ml o...

A concentrate of phosphosphingolipids from tobacco leaves has been resolved into two groups by chromatography on diethylaminoethylcellulose. The first group eluted contains no acetyl residues, whereas the second group contains one N-acetyl/mol of phosphorus. Three lipids from each group have been purified by chromatography on Porasil columns. The chemical composition of these six novel lipids shows them to be related to the previously characterized phosphosphingolipids PSL-I, N-acetylglucosamine[glucuronic acid, inositol phosphate]ceramide and PSL-II, glucosamine[glucuronic acid, inositol phosphate] ceramide [Kaul, K.,and Lester,R. L. (1975), Plant Physiol. 55, 120]. The novel lipids are: PSL-IA, PSL-I-[Ara2Gal2]; PSL-IB, PSL-I-[Ara3Gal2]; PSL-IC, PSL-I-[Ara4Gal2]; PSL-IIA, PSL-II-[Ara3Gal]; PSL-IIB, PSL-II-[Ara2or3Gal2]; PSL-IIC, PSL-II-[Ara2Gal2Man]. The unusual finding of unacetylated hexosamine in the PSL-II group of lipids appears not to be an artifact, as suggested by experiments involving rapid harvesting and processing of the tobacco leaves. Some of the fractions obtained by chromatography on diethylaminoethylcellulose appear to contain phosphosphingolipids with 16 or more sugar residues.

The chemical structure of a major glycophosphoceramide from tobacco leaves, called PSL-I [K. Kaul and R. L. Lester (1975), Plant Physiol. 55, 120], has now been characterized as 2-deoxy-2-acetamido-D-glucopyranosyl(alpha1 leads to 4)D-glucuronopyranosyl(alpha1 leads to 2)myoinositol-1-O-phosphoceramide. Sites of glycoside linkage were determined by (1) methylation analysis on a trisaccharide isolated by degradation of carboxyl-reduced PSL-I and (2) periodate oxidation experiments on PSL-I. The resulting products were identified with gas chromatography/mass spectrometry. Anomeric configurations were determined by resistance of the sugars in the peracetylated trisaccharide to chromium trioxide treatment.

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