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The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland.

ABSTRACT: 1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

SUBMITTER: White DA

PROVIDER: S-EPMC1183922 | biostudies-other | 1978 Mar

REPOSITORIES: biostudies-other

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Project description:1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.

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The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland.

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