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J. Biol. Chem., Vol. 268, Issue 3, 1702-1707, Jan, 1993

Purification and characterization of membrane-bound chitin synthase

S Machida and M Saito
National Food Research Institute, Ibaraki, Japan.

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.
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