Purification and characterization of 3'-phosphoadenylylsulfate: N-desulfoheparan sulfate sulfotransferase from arterial tissue.
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 beta-glucuronidase treatment. An N-desulfo-N-[1-14C]lacetylheparan sulfate deacetylase activity was copurified with the N-desulfoheparan sulfate sulfotransferase.
D000818:Animals; D001013:Aorta, Thoracic / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006497:Heparan Sulfate / Q000031:analogs & derivatives / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D013379:Substrate Specificity; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification* / Q000378:metabolism
D006497:Heparan Sulfate; D015238:Sulfotransferases; D013466:Sulfurtransferases; C040709:N-desulfoheparan sulfate; C022321:PAPS sulfotransferase