Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases

Assay methods were developed enabling separate determination of N- and O-sulphotransferase activities in an enzyme preparation from mouse mastocytoma. N-Desulphoheparin and chemically N-acetylated heparan sulphate were used as specific exogenous sulphate acceptors in the transfer of [³⁵S]sulphate residues from adenosine 3'-phosphate 5'-[³⁵S]sulphatophosphate to amino and hydroxyl groups respectively. The resulting ³⁵S-labelled polysaccharides were isolated as their cetylpyridinium complexes on filter paper. Sulphotransferases were solubilized from a mastocytoma microsomal fraction by treatment with detergent-alkali. The pH optimum for both enzymes was about 7.5 K_m with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 × 10^-5 M for the N-sulphotransferase and 1 × 10^-4 M for the O-sulphotransferase(s). The enzymes required bivalent cations for maximum activity, Mn²⁺ stimulating both the N- and O-sulphotransferase four- to five-fold, whereas Ca²⁺ increased the N- but not the O-sulphotransferase activity. The O-sulphotransferase was found to be more sensitive to heat-inactivation, 60% of the activity being lost after 1 min at 50 degrees C, whereas only 15% of the N-sulphotransferase activity was lost. In contrast, the N-sulphotransferase was selectively inhibited (or inactivated) by NaCl; at 0.125 M-NaCl concentration the O-sulphotransferase activity was essentially unaffected, whereas the N-sulphotransferase activity was depressed by 80%. These results strongly indicate that N- and O-sulphate-transfer reactions should be ascribed to different enzymes, or, alternatively, to separate and independent active sites on the same enzyme molecule.