caa a22 4500 397557469 CHVBK 20180308164806.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021293 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021293 Characterization of Chitin Synthase 2 of Saccharomyces cerevisiae II: Both Full Size and Processed Enzymes Are Active for Chitin Synthesis [Elektronische Daten] [Yukiko Uchida, Osamu Shimmi, Masayuki Sudoh, Mikio Arisawa, Hisafumi Yamada-Okabe] 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 chromatog-raphy, 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 36 kDa region encompassing the active site is sufficient for the catalytic activity. © by the Journal of Biochemistry Regular Papers nationallicence chitin synthase nationallicence product entrapment nationallicence Saccharomyces cerevisiae nationallicence zymogen nationallicence Uchida Yukiko Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut Shimmi Osamu Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut Sudoh Masayuki Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut Arisawa Mikio Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut Yamada-Okabe Hisafumi Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 659-666 0021-924X 119:4<659 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021293 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021293 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Uchida Yukiko Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut NATIONALLICENCE 700 1- Shimmi Osamu Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut NATIONALLICENCE 700 1- Sudoh Masayuki Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut NATIONALLICENCE 700 1- Arisawa Mikio Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut NATIONALLICENCE 700 1- Yamada-Okabe Hisafumi Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 659-666 0021-924X 119:4<659 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford