Purification and characterization of membrane-bound chitin synthase.
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.
Machida
S
S
National Food Research Institute, Ibaraki, Japan.
Saito
M
M
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002687:Chitin Synthase / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D004072:Digitonin; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D004795:Enzyme Stability; D005658:Fungi / Q000201:enzymology* / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D014357:Trypsin / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D002687:Chitin Synthase; D014357:Trypsin; D008274:Magnesium; D004072:Digitonin