Abstract
Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in beta-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 microM) and GlcNAc beta 1-6Man alpha 1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMc product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc beta-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosaminc could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.