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

L. Jansson, M. Hook, A. Wasteson, U. Lindahl

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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 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 activities 4 to 5 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°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.

Original languageEnglish (US)
Pages (from-to)49-55
Number of pages7
JournalBiochemical Journal
Volume149
Issue number1
DOIs
StatePublished - 1975

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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