Purification and characterization of membrane-bound chitin synthase

J Biol Chem. 1993 Jan 25;268(3):1702-7.

Abstract

The membrane-bound chitin synthase, a key enzyme of chitin biosynthesis, was purified, for the first time to homogeneity as a zymogen form. Digitonin could solubilize the enzyme from microsomal fraction of the filamentous fungus Absidia glauca, with 60-70% of the enzyme activity. The solubilized form of the enzyme was effectively purified by a sequence of chelating Sepharose, concanavalin A-Sepharose, and Mono Q column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave a single band with a molecular weight of 30,000. IgG prepared against this 30-kDa species on SDS-polyacrylamide gel electrophoresis immunoprecipitated chitin synthase. The purified enzyme existed as a zymogen, was converted into active form by treatment with trypsin, and the limited digestion with trypsin produced a little smaller polypeptide (28.5 kDa) of which the amino-terminal sequence was identical to the zymogen. The purified enzyme was the glycoprotein and showed a requirement for Mg2+. N-Acetylglucosamine stimulated the enzyme activity approximately 5-fold and polyoxin D, an analogue of substrate, and UDP, a byproduct of enzyme reaction, strongly inhibited the enzyme activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Membrane / enzymology
  • Chitin Synthase / chemistry
  • Chitin Synthase / metabolism
  • Chromatography, Gel
  • Digitonin
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Precursors / metabolism
  • Enzyme Stability
  • Fungi / enzymology*
  • Fungi / isolation & purification*
  • Immunosorbent Techniques
  • Magnesium / pharmacology
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Solubility
  • Trypsin / metabolism

Substances

  • Enzyme Precursors
  • Peptide Fragments
  • Chitin Synthase
  • Trypsin
  • Magnesium
  • Digitonin