Characterization of glucuronosyl transferase from Mucor rouxii: Requirement for polyuronide acceptors

Abstract

Most of the glucuronosyl transferase activity present in cell-free extracts from Mucor rouxii was found associated with a mixed membrane fraction (MMF). Treatment of this fraction with neutral detergents did not extract any transferase activity, but the insoluble enzyme became dependent on the addition of an exogenous primer, mucoric acid being more efficient than mucoran. Both polyuronides acted as true acceptors of glucuronosyl residues and were not merely enzymatic activators. Incubation of Triton-extracted MMF with UDP-glucuronic acid in the presence of UDP-GlcNAc, UDP-Glc, and UDP-Gal brought about a stimulation in the incorporation of glucuronic acid. This result was taken as evidence that, under these conditions, a heteropolymer was synthesized. Treatment with neutral detergents of native membrane fractions previously incubated with radioactive substrate extracted most of the incorporated radioactivity in the form of a polymer containing both protein and uronic acids. Treatment of this polymer with Pronase digested the protein, and radioactivity appeared in the form of low-molecular-weight compounds. Some properties of the glucuronosyl transferase treated with detergents were as follows: optimum temperature for enzyme activity was 26°C; the enzyme required a divalent metal ion, 4 m M MnCl 2 being the most efficient; and the enzyme showed biphasic kinetics with a Michaelis constant of 0.73 m M at low substrate concentrations.