Pulse-labelling of mouse
mastocytoma cell cultures, established from
ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled
heparin proteoglycan containing
polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular
polysaccharide fragments similar in size to commercially available
heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free
polysaccharide chains and, in addition, residual
proteoglycans that were of smaller size than the
proteoglycans initially pulse-labelled. The
polysaccharide chains released by
alkali treatment from the residual chase-incubated
proteoglycans were of the same size as the chains derived from
proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of
heparin proteoglycan to
polysaccharide fragments is initiated by release of intact
polysaccharide chains, probably by action of a
peptidase, and is pursued through cleavage of these chains by an
endoglycosidase. An
endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified
mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]
heparin proteoglycan to
polysaccharide fragments, but instead accumulated free intact
polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of
polysaccharide fragments was observed. It is tentatively proposed that the expression of
endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.