Abstract
The enzyme uridine diphosphate N-acetylglucosamine pyrophosphorylase was purified about 330-fold from an extract of baker's yeast by the treatment with protamine sulfate and column chromatographies on DEAF-cellulose, hydroxylapatite and Sephadex G-150. The purified enzyme was proved to be homogeneous by disc gel electrophoresis. The molecular weight was determined to be approximately 37, 000 by gel filtration. The enzyme had an optimum reactivity in the pH range of 7.5_??_8.5 and was stable at 4°C in potassium phosphate buffer, pH 7.5, containing 0.1mM dithiothreitol, but was unstable when stored at -20°C. The addition of dithiothreitol also increased the thermal stability of enzyme. The enzyme was specific for UDP-N-acetylglucosamine as substrate, and none of the other sugar nucleotides could serve as nucleotide substrate. The estimated values of Km were 6.1×10-3M for UDP-N-acetylglucosamine and 5.0×10-3M for inorganic pyrophosphate. The enzyme required some divalent cations for activity. Magnesium ion was the most effective among the cations tested. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol.
