Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate.
A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3'-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.
D000581:Amidohydrolases / Q000032:analysis*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D007700:Kinetics; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D014316:Tritium
D006025:Glycosaminoglycans; C527794:N-acetylheparosan; D011135:Polysaccharides, Bacterial; D014316:Tritium; D000581:Amidohydrolases; C040499:N-acetylheparosan deacetylase