Expression and Characterization of Endopeptidase in Suspension-cultured Cells of French Bean

Abstract

When suspension cells of French bean (Phaseolus vulgaris L. cv. Goldstar) were cultured in Murashige and Skoog (MS) liquid medium containing 10⁻⁶ m 1-naphthaleneacetic acid (NAA) and 10⁻⁶ m 6-benzylaminopurine (BAP) (control medium), endopeptidase activity was expressed throughout the cell growth. In the medium containing 10⁻⁵ m gibberellic acid (GA₃), the endopeptidase activity increased notably during the cell culture, but in the medium containing amino acids the activity decreased. Protein immunoblotting with an antiserum raised against a French bean cysteine endopeptidase (EP-C1) showed that a 34-kDa polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from cultured cells. Five forms of endopeptidase in extracts of cells cultured in the control medium and the four forms in the medium containing GA₃ were detected by activity staining after non-denaturing polyacrylamide gel electrophoresis. Experiments with various protease inhibitors indicated that a serine endopeptidase was expressed at high levels in cultured cells in the control medium and the activity of a cysteine endopeptidase was increased by GA₃.

Key words:

• endopeptidase
• gibberellic acid
• Phaseolus vulgaris
• protease; suspension cells