Very complex glycosphingolipids with A, H and I blood-group activities were isolated from human erythrocyte membranes. The membranes were obtained from erythrocytes of blood group A, A2 a n d 0 respectively. A general formula for the antigens is:(where Fuc is fucose, Gal is galactose, GlcNAc is N-acetylglucosamine and Glc is glucose) with values of y2 ranging from 10-27. A-active preparations contain additionally 2-3 residues of N-acetylgalactosamine. In view of the unusual complexity of these compounds they were designated poly(glycosy1)ceramides (formerly megaloglycolipids). Individual poly(glycosy1)ceramide fractions were isolated from A erythrocytes and were found to differ by about 8 glycosyl residues per molecule forming a series of compounds with 22, 30, 38, 51 and 59 glycosyl residues per mole. Structural studies indicate that the main sequence of poly(glycosy1)ceramides consists of the residues of galactopyranose and 2-deoxy-2-acetamidoglucopyranose substituted at 3 and 4 position respectively. These residues are probably alternating. N-Acetylglucosamine substituted at 3 position was not found in poly-(glycosy1)ceramides. Branches of poly(glycosy1)ceramides originate from 3 and 6 position of galactopyranosyl residues. The number of branches is proportional to the degree of molecular complexity. In poly(glycosy1)ceramides isolated from A and A2 erythrocytes the branches are terminated with the following structures GalNAc CI 1 +3 [Fuc CI 1 '21 Gal; Fuc CI 1 -i2 Gal and Gal (presumably Gal 1 -+4 GlcNAc). In poly(glycosy1)ceramides from A cells the total number of A and H-active structures per average molecule of 30-35 glycosyl residues amounts to 2.1 and 1.2 respectively while the number of terminal galactose structures is 1.8. For poly(glycosy1)ceramides from A2 erythrocytes the corresponding figures are 0.75, 3.5, and 2.1 respectively.Poly(glycosy1)ceramides from 0 cells comprise about 3.8 H-active structures and 1.8 terminal galactopyranosyl residues. In poly(glycosy1)ceramides with high "n" values the number of terminal galactose structures is increased. These fractions display high blood-group I activity. However, the removal of terminal galactose with P-galactosidase affects I-activity only slightly.

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosy1)ceramides. In contrast to poly(glycosy1)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosy1)ceramides.Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transferase of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAcl + 6Gal structures.

Fucose-containing glycolipids with H and B blood group activity as well as sialic acid and glucosamine-contaig glycolipid were isolated from human erythrocytes. The sequences and linkages of carbohydrate residues in these glycolipids were determined by partial acid hydrolysis, periodate oxidation and methylation studies. Anomeric linkage were ascribed to those materials on the basis of their optical rotations, infrared spectra, susceptibility to enzymes and immunological activities.

The isolation and purification of new group A-, B-and H-specific substances from human erythrocyte membranes is described. These substances contain galactose, fucose, glucosamine, sialic acid and amino acid residues. The A-specific substance also contains galactosamine. They contain a t most about Z0I0 fatty acids or sphingosine. The substances are serologically active; however, both required combination with phospholipids for development of maximum activity ine haemagglutination inhibition tests. Addition of phospholipids was not required in serological precipitation tests. These blood-group substances may be used to coat group-0 or Bombay-Oh red cells, thereby conferring on them A-, B-, or H-activity. They form high-molecular-weight aggregates in aqueous solution but disaggregate into smaller units in the presence of sodium dodecylsulphate.The glycolipid nature of A-and B-antigens in human erythrocytes was established in the last decade [1,2] However some recent evidence obtained in different laboratories [lo-141 indicated that glycoprotein substances specific for blood-group A, B and H occur in human erythrocytes as well as glycolipids. This paper presents an attempt to purify and characterise immunologically and chemically these recently discovered substances. A Preliminary account of this work was presented in an abstract form [15].Enzymes. a-l,2-~-E'ucosidase (EC 3.2.1.-) ; a-N-acetylgalactosaminidase (EC 3.2.1.-); B-galactosidase (EC 3.2.1.23). MATERIALS AND METHODS Preparation of Erythrocyte StromaErythrocyte stroma were prepared from the outdated blood by the toluene flotation technique of Kobcielak et al. [3]. The dried stroma were extracted with 83O/, ethanol and the extract cooled and centrifuged to remove the active glycolipids [16]. Half of the volume of the supernatant was then warmed to room temperature and used for reextraction of the stroma powder left after the first ethanol extraction. This procedurc was repeated once more, the dissolved glycolipids being removed each time by cooling and centrifuging the extract. The final yield of ethanol-extracted stroma ranged from 20 t o 25 g per liter of packed erythrocytes. Xerological AssaysHaemagglutination inhibition tests were performed by the method of Morgan and King [17]. Normal human anti-A sera were used for the determination of A-activity. For the determination of H-activity the sera were prepared by immunisation of rabbits with human-cyst H-substance coupled to the protein component of somatic-0 antigen of Xhigella

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