Incubation of a mouse mastocytoma microsomal fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded proteoglycans containing non-sulphated polysaccharide chains. Similar incubations performed in the presence of sulphate donor 3′-phosphoadenosine 5′-phosphosulphate (PAPS) produced both sulphated and non-sulphated proteoglycans, which were separated by chromatography on DEAE-cellulose Analysis by gel chromatography of single polysaccharide chains, released from the proteoglycans by alkali treatment, showed that the non-sulphated chains produced during incubation for 5 min or 25 min, either in the absence or in the presence of PAPS, were of fairly small molecular size, with an average peak Mr of approx. 10 x 10(3)-15 x 10(3). In contrast, the sulphated chains exceeded Mr 100 x 10(3) Pulse-chase experiments suggested that sulphated chains were capable of further elongation. These results indicate that sulphation promotes, by so far unknown mechanisms, further chain elongation. Sulphated proteoglycan (retarded on DEAE-cellulose chromatography) isolated after similar incubation of the microsomal fraction for 1 min only was found to contain a mixture of sulphated and virtually non-sulphated polysaccharide chains. However, when [35S]PAPS was included in the incubations, some 35S was found to be associated, essentially as N-sulphate groups, also with the latter type of chains, preferentially the high-Mr fraction. These results are interpreted in terms of a biosynthetic model by which the heparin proteoglycan is generated through transient interactions of macromolecular intermediates with distinctly separate complexes of membranebound enzymes.

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