Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.
When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.
Uchida
Y
Y
Department of Mycology, Nippon Roche Research Center, Kanagawa.
Shimmi
O
O
Sudoh
M
M
Arisawa
M
M
Yamada-Okabe
H
H
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D002686:Chitin / Q000096:biosynthesis; D002687:Chitin Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002918:Chymotrypsin; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000276:immunology; D017384:Sequence Deletion; D014357:Trypsin
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D002686:Chitin; D002687:Chitin Synthase; D002918:Chymotrypsin; D014357:Trypsin