Biosynthesis of heparin. Concerted action of early polymer-modification reactions.
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In a previous report on the biosynthesis of heparin, an N-acetyl-D-glucosaminyl N-deacetylase present in a mouse mastocytoma microsomal fraction was characterized by its ability to release [3H]acetate from an exogenous, N-[3H]acetyl-labeled, nonsulfated heparin-precursor polysaccharide. Exhaustive incubation with repeated additions of fresh enzyme led to release of only 30 to 35% of the [3H]acetyl groups originally present in the polysaccharide. In the present communication, additions of 3'-phosphoadenylyl sulfate (PAPS) to incubation mixtures are shown to markedly increase the release of [3H]acetate. Maximal effect, a 3- to 4-fold increase in reaction rate, was obtained at 0.1 mM PAPS concentration. The stimulatory effect of PAPS is due to modification (N-sulfation) of the substrate molecules rather than activation of the deacetylating enzyme, as suggested by the following observations. Polysaccharide substrate was subjected to exhaustive incubation with microsomal enzyme, in the absence of PAPS. The resulting product, essentially resistant to further N-deacetylation, could be reconverted into a substrate for the N-deacetylase, not only by acetylation but also, and more efficiently, by chemical sulfation of the exposed free amino groups. Furthermore, incubation of exogenous, nonsulfated polysaccharide with microsomal fraction in the presence of PAPS resulted in extensive incorporation of sulfate groups, approaching that observed after sulfation of endogenous, microsomal polysaccharide. In the most highly sulfated fraction, more than half of the acetamido groups had been replaced by sulfamido groups, but not to any significant extent by unsubstituted amino groups. The concerted action of the N-deacetylase and N-sulfotransferase enzymes may constitute an important regulatory event in the biosynthetic polymer-modification process.
author list (cited authors)
Riesenfeld, J., Höök, M., & Lindahl, U
complete list of authors
Riesenfeld, J||Höök, M||Lindahl, U