Biosynthesis of Heparin

The biosynthesis of the neutral trisaccharide, 3-O-β-d-galactosyl-4-O-β-d-galactosyl-d-xylose of the heparin-protein linkage region, has been investigated. A microsomal fraction of heparin-producing mouse mastocytoma catalyzed the incorporation of galactose from UDP-galactose into endogenous as well as exogenous substrates. Partial acid hydrolysis of 14C-galactose-labeled endogenous acceptor yielded several fragments with the characteristics of neutral oligosaccharides previously isolated from the heparin-protein linkage region and, in addition, a compound with the chromatographic properties of N-acetyllactosamine. The stepwise synthesis of the neutral trisaccharide was investigated with separate assays for the two galactosyl transferase reactions, with exogenous acceptors such as d-xylose (product, 4-O-β-d-galactosyl-d-xylose) and 4-O-β-d-galactosyl-d-xylose (product, 3-O-β-d-galactosyl-4-O-β-d-galactosyl-d-xylose). Treatment of the particulate enzyme with ammonium hydroxide in the presence of Tween 20 resulted in the solubilization of considerable amounts of galactosyltransferase activities. Whereas only 4% of the UDP-galactose-d-xylose galactosyltransferase was brought into solution by treatment of the enzyme with detergent at neutral pH, about one-third of the transferase activity related to the synthesis of the second galactose moiety was recovered in the supernatant under similar conditions, indicating that the two reactions are catalyzed by two different enzymes. A third galactosyltransferase which catalyzed the transfer of galactose to N-acetylglucosamine was also detected in the microsome particles. This enzyme was solubilized by the same procedure as that used for the two transferases mentioned above but was discriminated from the latter by its stability toward heat and by fractionation with ethanol.