Blood‐Group ABH‐Specific Macroglycolipids of Human Erythrocytes: Isolation in High Yield from a Crude Membrane Glycoprotein Fraction

Highly glycosylated, water‐soluble ABH‐specific sphingolipids, designated macroglycolipids, were isolated in high yield, up to 5 mg per unit of blood, from the crude human‐erythrocyte‐membrane glycoprotein fraction which is obtained by extraction of the membranes with chloroform/methanol/ water. Both serological tests and radioactive labelling experiments indicated that these substances, rather than the glycoproteins, are the principal ABH‐components in this fraction. The activities of A‐specific, B‐specific and H‐specific macroglycolipids were very high, approximately 0.1 M̈g inhibiting four hemagglutinating doses of the respective agglutinating reagents, and were thus comparable to those of secreted blood‐group ABH‐specific glycoproteins. The substances were stable to mild alkaline conditions. They contained fucose, galactose, glucosamine, glucose, sialic acid, sphingosine and fatty acids; blood‐group‐A‐specific substances contained, in addition, galactosamine. No amino acids were detected. Assuming one glycosyl residue per molecule, the average number of sugars in A and B macroglycolipids was 31, and their molecular weights approximately 6100. The presence of β‐D‐galactosidase‐labile and sialic acid residues indicated that these substances contain nonreducing termini additional to the ABH immunodeterminants. In the B macroglycolipid, the ratio between nonreducing terminal α‐D‐galactopyranosyl and β‐D‐galacto‐pyranosyl residues was 1.7:1.0. The macroglycolipids formed clear aqueous solutions at concentrations as high as 30 mg/ml, were insoluble in 60–70% aqueous ethanol, and did not migrate on thin‐layer chromatography unless they were acetylated. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl‐sulfate showed the macroglycolipids to be a heterogeneous mixture migrating throughout most of the region in which the periodic acid/Schiff‐positive membrane glycoproteins are found. On the basis of the evidence presented, it is concluded that macroglycolipids are the predominant ABH‐specific component in human erythrocyte membranes, and that they most likely account for previous observations of ABH activity in membrane glycoprotein fractions.