Purification and characterization of 3′‐phosphoadenylylsulfate: N‐desulfoheparan sulfate sulfotransferase from arterial tissue

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Abstract
A 3′‐phosphoadenylsulfate: N‐desulfoheparan sulfate sulfotransferase (EC 2.8.2.12) was purified 450‐fold from the microsomal fraction of calf arterial tissue and separated from 3′‐phosphoadenylylsulfate: chondroitin sulfotransferase (EC 2.8.2.5) activity.The enzyme has optimal activity at neutral pH, requires divalent cations (Mn2+, Mg2+, Ca2+) for maximal activity and exhibits specificity towards N‐desulfoheparan sulfate, N,O‐desulfoheparan sulfate and oligosaccharides derived therefrom. N,O‐desulfoheparan sulfate tetrasaccharides serve as acceptor substrates only if the nonreducing terminus is occupied by glucuronic acid (not iduronic acid). The N,O‐desulfoheparan sulfate sulfotransferase transfers [35S]sulfate from 3′‐phosphoadenylyl[35S]sulfate to the 2‐amino groups and to the 6‐hydroxy groups of glucosamine units of the acceptor substrates. The ratio of N/O‐sulfation ranged between 3:1 and 2:1. O‐[35S]Sulfated unsaturated disaccharides were obtained from enzymatically labelled [35S]N‐desulfoheparan sulfate by heparitinase degradation and subsequent deamination. Evidence for the O‐sulfation at C‐6 of the glucosamine units was provided by isolation of anhydromannose [35S]monosulfate, which was formed from uronosylanhydromannose [35S]monosulfate by β‐glucuronidase treatment. An N‐desulfo‐N‐[1‐14C]acetylheparan sulfate deacetylase activity was copurified with the N‐desulfoheparan sulfate sulfotransferase.