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Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose.

ABSTRACT: UDP-galactose appears to be produced on one side of a membrane barrier, opposite the galactosyltransferases that use it as a sugar donor. The translocation of activated galactose across membranes was studied in rat submaxillary-gland microsomal vesicles and in rat liver Golgi vesicles. When these intact vesicles containing the acceptor, N-acetylglucosamine, were incubated in the presence of UDP-galactose and two inhibitors of galactosyltransferase activity, the product, N-acetyl-lactosamine, formed within the vesicles. Thus at least the galactose moiety of UDP-galactose crossed the membranes. When intact Golgi vesicles were incubated with UDP-galactose labelled in both the uridine and the galactose moieties, labelled N-acetyllactosamine was again produced in the vesicles, but less than stoichiometric amounts of the uridine label was found there. Calculation of internal and external concentrations of UMP, a major product released from the cleaved uridine moiety, showed that the vesicles were actually enriched in UMP. When free UMP was incubated with the vesicles, this enrichment did not occur. This result was direct evidence for facilitated transport of UDP-galactose into the Golgi for use by galactosyltransferase.

SUBMITTER: Barthelson R

PROVIDER: S-EPMC1144554 | biostudies-other | 1985 Jan

REPOSITORIES: biostudies-other

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Project description:An enzyme which catalyses the transfer of sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcalpha2-3Galbeta1-4GlcNAc (3'SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3'-sialyl-6'-sulpho-LacNAc [NeuAcalpha2-3Gal(6-SO4)beta1-4GlcNAc] and 3'-sialyl-6'-sulpho-LewisX [NeuAcalpha2-3Gal(6-SO4)beta1-4(Fucalpha1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an alpha2-3-linked sialic acid residue, although GlcNAc in 3'SLN can be substituted by Glc, and Galbeta1-4GlcNAc can be replaced by Galbeta1-3GlcNAc without loss of activity. The finding that 3'-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcalpha2-3Galbeta1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3'SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra) bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3'SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12-25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 microM for PAPS, and 0.72 mM and 1.16 mM for 3'SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3'SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

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Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose.

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