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 Km with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 X 10(-5) M for the N-sulphotransferase and 1 X 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.
D002413:Cations, Divalent; D002460:Cell Line; D006493:Heparin / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship; D013466:Sulfurtransferases / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D002413:Cations, Divalent; D006493:Heparin; D006497:Heparan Sulfate; D011189:Potassium Chloride; D012965:Sodium Chloride; D013466:Sulfurtransferases