Taro Hori

Studies on sphingolipids of fresh-water mussel spermatozoa

Abstract 1. 1. The total lipid was extracted from the spermatozoa of fresh-water mussel ( Hyriopsis schlegelii ) with chloroform-methanol and the extract was treated with mild alkali. The alkali-stable lipid fraction,which mainly included the sphingolipids, was fractionated on a silicic acid column. 2. 2. Five classes of sphingolipid were separated and characterized as an unidentified sphingolipid, ceramide monohexoside, ceramide 2-aminoethylphosphonate and two kinds of mucolipids. The ceramide monohexoside was composed of ceramide galactoside and a trace amount of ceramide glucoside. No sphingomyelin was found in the sphingolipids. 3. 3. The sugar moieties of the mucolipids were composed of fucose, xylose, mannose and glucose in a ratio of 1:1:2:3 and the amino sugar of the mucolipids was identified as glucosamine, while no sialic acid was detected in them. 4. 4. The fatty acid compositions of the sphingolipids were mainly composed of saturated acids, in which palmitic and stearic acids were predominant.

1H-2D-nuclear magnetic resonance applied to the primary structure determination of a novel octasaccharide glycolipid isolated from the spermatozoa of bivalves

High resolution, two-dimensional 1H-n.m.r. spectroscopy has been used to confirm a proposed primary structure of a glycolipid having an octasaccharide head-group. Pure absorption and relay experiments were found to be particularly useful in establishing connectivities in poorly resolved regions of the spectrum. The spectral assignments, which indicate novel linkages including an internal fucopyranosyl residue as well as terminal xylosyl and 4-O-methylglucopyranosyluronic acid groups, add to a growing data base for structural characterization through n.m.r. spectroscopy.

Branched long-chain bases from the bivalve corbicula sandai

Long-chain bases were liberated from a crude mixture of sphingolipids from whole tissue of the fresh-water bivalve C. sandai, and conversion of the bases into N-acetyl-0-trimethylsily derivatives was accomplished. The derivatized bases were analyzed by combined gas-liquid chromatography and mass spectrometry. A portion of the sphingolipids was subjected to catalytic hydrogenation from whch saturated long-chain bases (sphinganines) were obtained. The saturated bases were oxidized with lead tetra-acetate and the aldehydes produced were analyzed by gas-liquid chromatography. The aldehydes were further oxidized to acids with silver oxide, the resulting fatty acids methylated and also analyzed by gas-liquid chromatography. By these analyses, altogether five long-chain bases were identified, consisting of hexadeca-4-sphingenine (15%), heptadeca-4-sphingenine (2%), iso-octadeca-4-sphingenine (13%), octadeca-4-sphingenine (39%) and anteiso-noadeca-4-sphingenine (31%). So far no branches have been found in shellfish spingolipid long-chain bases.

Biochemistry of shellfish lipids XI. Incorporation of [32P]orthophosphate into ceramide ciliatine (2-aminoethylphosphonic acid) of the fresh-water mussel, Hyriopsis schlegelii

Abstract 1. 1. Thin-layer chromatography was employed to demonstrate that 32 P i was incorporated in vivo into ceramide ciliatine of the fresh-water mussel, Hyriopsis schlegelii . 2. 2. Evidence was shown that the incorporation was most effective in liver where it accounted for 16% of the phospholipid fraction. 3. 3. Label incorporation into ciliatine from 32 P i was achieved with a liver homogenate.

Di- and trimannosylceramides in hepatopancreas of a fresh-water bivalve, Hyriopsis schlegelii

An investigation of di- and triglycosylceramides of hepatopancreas of a fresh-water bivalve, Hyriopsis schlegelii, gave the following results: (1) The total amount of neutral glycosphingolipid was 7.2 mg/g of the dry weight of the hepatopancreas. (2) The diglycosylceramide fraction accounted for 4.8% of the total neutral glycolipid, and consisted of Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer, Man (beta 1 leads to 4)Glc leads to Cer, Gal(beta 1 leads to 4)Glc leads to Cer, and Gal(beta 1 leads to 4)Gal(beta 1 leads to 1)Cer. The presence of so many different types of diglycosylceramides in a single tissue appears to be the exception rather than the rule. (3) The triglycosylceramide fraction amounted to 12.2% of the total neutral glycolipid and contained a mixture of Man(alpha 1 leads to 3)Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer and Man(alpha 1 leads to 3)Man(beta 1 leads to 4)Glc leads to Cer. (4) The spectrum of di- and triglycosylceramides of the hepatopancreas was more complex than that of spermatozoa of this bivalve. (5) It is an interesting finding that a series of glycolipids containing one to three mannose residues occurs in nature. (6) The ceramide moieties of the above two fractions contained the normal saturated fatty acids in the range of C14-C24, and derivatives of 4-sphingenine. The structural analysis involved (1) gas-liquid chromatography of the component sugars, fatty acids and long-chain bases; (2) methylation studies coupled with gas-liquid chromatography and mass spectrometry to locate the bonds between the hexose units; and (3) enzymatic degradation to establish the sugar sequence and the anomeric configuration of the glycosidic links.

Characterization of a novel ceramide octasaccharide isolated from whole tissue of a fresh-water bivalve, Corbicula sandai

A novel glycosphingolipid containing two mannose residues, provisionally named GL-1 in a previous paper (Itasaka, O. and Hori, T. (1979) J. Biochem. 85, 1469-1481), was isolated from whole tissue of a fresh-water bivalve, Corbicula sandai, and the structure of the glycolipid was determined to be: Galp4Me(beta1 more than 3)GalpNAc(beta1 more than 3)Fucp(alpha1 more than 4)GlcpNAc(beta1 more than 2)Manp(alpha1 more than 3)(Xylp(alpha1 more than 2))Manp(beta1 more than 4)Glcp(beta1 more than 1)Cer. Glycosphingolipid-1 seems to be a biosynthetic precursor of glycosphingolipid-3, because they have an identical octasaccharide structure, except for the presence of an aminoethylphosphoryl residue in glycospingolipid-3. However, in contrast to glycosphinogolipid-3, glycosphingolipid-1 has a more complex ceramide pattern. This fact suggests that glycosphingolipid-1 as isolated is not a degradation product of glycosphingolipid-3, and it can be presumed that the restricted molecular species of glycosphingolipid-1 which has the same ceramide constitution as glycosphingolipid-3 may be utilized preferentially for glycosphingolipid-3 biosynthesis.

Identification of β-d-mannosylceramide in hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii

A mannosylceramide was isolated by preparative thin-layer chromatography on a 3% borate-impregnated silica gel plate from a monohexosylceramide fraction of the hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii. It contained only mannose as the sugar component and the ceramide moiety contained mainly sphingosine and palmitic acid. Anomeric configuration of the sugar moiety was determined by enzymatic hydrolysis with beta-D-mannosidase. The concentration of this glycolipid was 5% of the total monohexosylceramide fraction of the hepatopancreas.

Chapter 3 Phosphonolipids

Publisher Summary This chapter discusses phosphonolipids. The combined use of diethylaminoethyl (DEAE)-cellulose and silicic acid column chromatography has been effective in the isolation of phosphonolipids. A mixture of phosphonolipids and phosphatidylethanolamine can be recovered from a DEAE-cellulose column by elution with chloroform–methanol (7:3). Their further separation can be achieved by silicic acid column chromatography. For the isolation of SPnL from the tissues of mollusks and coelenterates, the lipid extracts obtained with chloroform–methanol are subjected to mild alkaline hydrolysis to remove glycerolipids; the alkali-stable lipid fraction is fractionated by silicic acid column chromatography using increasing proportions of methanol in chloroform as solvent. The individual phosphonolipids are finally purified by preparative thin-layer chromatography. Nuclear magnetic resonance (NMR) provides the ideal method for direct detection and determination of phosphonolipids. The absorption band of phosphonolipids is well-separated from the band of phospholipids in the NMR spectra of intact lipids by [ 31 P]NMR. The amounts of the phosphonolipids, determined from the respective areas of the absorption bands in each spectrum are identical to those determined by the classical colorimetric procedure for the quantitation of phosphonate phosphorus.