J. Biochem, 1996, Vol. 119, No. 4 659-666
© 1996 Japanese Biochemical Society
research-article |
Characterization of Chitin Synthase 2 of Saccharomyces cerevisiae II: Both Full Size and Processed Enzymes Are Active for Chitin Synthesis
Department of Mycology, Nippon Roche Research Center 200 Kajiwara, Kamakura, Kanagawa 247
1;To whom correspondence should be addressed. Tel: + 81-467-47-2213, Fax: +81-467-46-5320, E-mail: hisafumi.okabe{at}roche.com
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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Broehan, L. Zimoch, A. Wessels, B. Ertas, and H. Merzendorfer A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm J. Exp. Biol., October 15, 2007; 210(20): 3636 - 3643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Zhang, R. Kashimshetty, K. E. Ng, H. B. Tan, and F. M. Yeong Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to mother-daughter neck via the secretory pathway in budding yeast J. Cell Biol., July 17, 2006; 174(2): 207 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Merzendorfer and L. Zimoch Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases J. Exp. Biol., December 15, 2003; 206(24): 4393 - 4412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Trilla, A. Duran, and C. Roncero Chs7p, a New Protein Involved in the Control of Protein Export from the Endoplasmic Reticulum that Is Specifically Engaged in the Regulation of Chitin Synthesis in Saccharomyces cerevisiae J. Cell Biol., June 14, 1999; 145(6): 1153 - 1163. [Abstract] [Full Text] [PDF]
|
|