Biosynthesis of heparin. Partial purification of the uronosyl C-5 epimerase.

Abstract

Heparosan N-sulfate D-glucuronosyl 5-epimerase, which catalyzes the conversion of beta-D-glucuronosyl to alpha-L-iduronosyl residues in the course of heparin biosynthesis, has been purified approximately 9000-fold from the high speed supernatant fraction of a homogenate of a mouse mastocytoma. Following ammonium sulfate fractionation, the material precipitating between 35 and 60% saturation was subjected to a series of affinity chromatography steps on matrices containing immobilized concanavalin A, heparan sulfate, O-desulfated heparin, and Cibacron blue, respectively. Epimerase purified by this procedure yielded two major components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had approximately the same Kav as bovine serum albumin when chromatographed on Sepharose 6B. The activity of the purified enzyme was increased 50-fold by addition of the fraction which was not adsorbed to concanavalin A-Sepharose. The stimulating factor is likely to be a protein since it was nondialyzable, heat labile, and lost activity on digestion with trypsin.