Abstract
A novel glycosphingolipid, provisionally named Lipid IV (Hori, T., Sugita, M., Ando, S., Kuwahara, M., Kumauchi, K., Sugie, E., and Itasaka, O. (1981) J. Biol. Chem. 256, 10979-10985), was obtained from spermatozoa of the fresh water bivalve, Hyriopsis schlegelii. The yield of the glycolipid was 2.2 mg/g of dried spermatozoa. The structure of the glycolipid was elucidated by partial hydrolysis, permethylation analysis, and proton nuclear magnetic resonance. The following structure is proposed: GlcA4Me beta 1-4(GalNAc3Me alpha 1-3)Fuc alpha 1-4GlcNAc beta 1-2Man alpha 1-3(Xyl beta 1-2)Man beta 1-4Glc beta 1-Cer. The acidic glycolipid is unique in containing 4-O-methylglucuronic acid as well as an internally located fucose. Palmitic acid, stearic acid, and C18-sphingosine are the major aliphatic components. This composition is similar to those of other neutral spermatozoan glycolipids, Lipid I (Hori, T., Sugita, M., Kanbayashi, J., and Itasaka, O. (1977) J. Biochem. (Tokyo) 81, 107-114) and Lipid II (Hori, T., Takeda, H., Sugita, M., and Itasaka, O. (1977) J. Biochem. (Tokyo) 82, 1281-1285), suggesting a possible metabolic relationship among them.
Highlights
The yield of the glycolipid was 2.2 mg/g of dried spermatozoa
Inthisstudy we have isolateda novel acidic glycolipid (Lipid IV),containing4-0-methylglucuronicacid,from H . schlegelii spermatozoa. This is the first case of a glycolipid having a 4-0-methylglucuronic acid residue, while 4-0-methylglucuronic acid has been found in the capsularpolysaccharides from Klebsiella typex [5],birch xylan [6],and hardwood xylan [7]
Isolation of Lipid IV-The total glycosphingolipid fraction was obtainedfrom lyophilized spermatozoa (100 g) by extractionwith chloroform/methanol (2:1, I:I, and 1:2, v/v), mild alkaline hydrolysis, and Unisil columnchromatography [8] as reported previously [2]
Summary
Materials-The materials used were essentially the same as described in the previous work [4]. Isolation of Lipid IV-The total glycosphingolipid fraction was obtainedfrom lyophilized spermatozoa (100 g) by extractionwith chloroform/methanol (2:1, I:I, and 1:2, v/v), mild alkaline hydrolysis, and Unisil columnchromatography [8] as reported previously [2]. Theupperaqueous phase was concentratedtonear dryness and redissolved in 25 ml of chloroform/methanol (1:1,v/v) and applied to a DEAE-Sephadex A-25 (acetate form) column The column was washed with 400 ml of methanol, and acidic lipids were eluted with a linear gradient of increasing concentrations of ammoniumacetate (0.05 to 0.20 M) in methanol(total volume, 700 ml) [11].Each 5 ml was collected, and the concentration of uronic acid in the fractions was determined by the colorimetric method using carbazol-HaS04reagent [12]. L'he neutral lipid fraction eluted from the columnwith thesame solvent was subjected to Iatrobeads column chromatography using a linear gradient system, chloroform/methanol/water(1kIO:l to 10405,v/v). A constant flow rate of 30 ml/h was attained by a DCL microconstametricpump
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