Incorporation of 14c and 3h from Nucleotide Sugars into a Polysaccharide in the Presence of a Cell-free Preparation from Mouse Mast Cell Tumors.

The incorporation of glucose-% and 35S-labeled inorganic sulfate into heparin has been demonstrated in tissue slices of mouse mast cell tumors (1, 2). In addition, incorporation of 35S-labeled inorganic sulfate has been demonstrated in cell-free systems of DBA mouse mast cell tumors with 3’-phosphoadenosine 5’-phosphosulfate as a probable intermediate (3-6). However, the enzymatic reactions leading to the formation of the polysaccharide chain of heparin are not known, nor have the steps been elucidated at which amino and ester sulfation occur. The incorporation of glucuronic acid-% from uridine diphosphate (UDP) glucuronic acid-14C and glucosamine-14C from UDP-N-acetylglucosamine-‘4C into a material which appeared to be a polysaccharide was reported in a preliminary communication (7). The reaction proceeded in the presence of a microsomal preparation from heparin-synthesizing DBA mouse mast cell tumors, and required both UDP-glucuronic acid and UDP-Nacetylglucosamine for optimal incorporation. UDP-glucosamine could not be substituted for UDP-N-acetylglucosamine. The data in the present report indicate that there is simultaneous and equimolar uptake of glucuronic acid-l4C from UDPglucuronic acid-14C and acetyl-3H from UDP-N-acetyl-3H-glucosamine into polysaccharide in the presence of a microsomal preparation from DBA tumors. Uptake of glucosamine-lC from UDP-N-acetylglucosamine-‘4C has also been demonstrated in the presence of UDP-glucuronic acid. The hydrolysate of the 14C-labeled polysaccharide formed from UDP-N-acetylglucosamine%! has been found to have glucosamine as its only labeled sugar. The radioactive polysaccharide has many of the characteristics of hyaluronic acid. However, it is resistant to degradation by testicular hyaluronidase, but susceptible to degradation by bacterial heparinase. It is postulated that this is a nonsulfated, acetylated polysaccharide which may be a heparin precursor.