Biosynthesis of heparin. Assay and properties of the microsomal N-acetyl-D-glucosaminyl N-deacetylase.

In a previous study on heparin biosynthesis, incubation of a mouse mastocytoma microsomal fraction with UDP-N-acetyl-D-glucosamine and UDP-D-glucuronic acid resulted in the formation of two nonsulfated polysaccharides, one of which was fully N-acetylated whereas the other contained about equal amounts of Nacetylated and N-unsubstituted glucosamine residues. In the presence of 3’-phosphoadenylyl sulfate the latter species was converted into heparin through a series of reactions initiated by sulfation of the unsubstituted amino groups (Hook, M., Lindahl, U., Hallen, A., and Backstrom, G . (1975) J. Biol. Chem 250,6065-6071). Incubation of the fully N-acetylated polysaccharide with microsomal enzyme resulted in partial N-deacetylation, as shown by the formation of a product susceptible to deamination with nitrous acid. Characterization of the deamination products by gel chromatography suggested that the N-acetyl groups had been attacked in a random fashion. An assay for the Ndeacetylase was developed, based on the liberation of labeled acetate from a N-[3H]acetylated heparin precursor polysaccharide; the released C3H]acetate was recovered by extraction with ethyl acetate and quantified by liquid scintillation counting. The rate of acetate release was linear with time and directly proportional to the concentration of microsomal enzyme. The reaction required Mn2+ ions and had a pH optimum between 6.0 and 6.5. Salts were inhibitory at relatively low concentration (50% inhibition at 50 m~ NaCl concentration). Exhaustive incubation of the labeled substrate with microsomal enzyme caused release of 30 to 35% of the [3H]acetyl groups originally present; this value could be increased only by reincubating the substrate after substitution of the N-unsubstituted D-glucosamine residues with (unlabeled) acetyl groups. Incubation of various labeled glycosaminoglycans with mastocytoma microsomal fraction showed that only polysaccharides structurally related to heparin (microsomal heparin-precursor polysaccharides, and, in addition, a N-desulfated, N-acetylated heparan sulfate) were susceptible to N-deacetylation. Other N-acetylated polysaccharides, such as hyaluronic acid, chondroitin sulfate, and dermatan sulfate were not attacked by the N-deacetylase.