beta-Amylase from Mustard (Sinapis alba L.) Cotyledons : Immunochemical Evidence for Synthesis de Novo during Photoregulated Seedling Development

Abstract

A polyclonal antiserum against mustard (Sinapis alba L.) beta-amylase was obtained by injecting a homogeneously purified enzyme preparation in rabbits. The formation of beta-amylase specific antibodies was confirmed by staining the precipitin line in double diffusion gel for beta-amylase activity. The monospecificity of antiserum against mustard beta-amylase was also ascertained by Western blotting. The antiserum efficiently recognised both the denatured and the native form of beta-amylase, but it did not cross-react with other higher plant beta-amylase. The mode of photoregulation of beta-amylase activity in mustard cotyledons was investigated by a variety of immunochemical techniques. Immunotitration experiments ruled out the possible contribution of enzyme activation/inactivation in photoregulation of beta-amylase activity. The use of single radial immunodiffusion, rocket immunoelectrophoresis, and immunotitration confirmed that the light mediated increase in beta-amylase activity quantitatively corresponds with the increase in beta-amylase protein level. The in vivo labeling with l-[(35)S] methionine and pulse chase studies of in vivo labeled beta-amylase protein revealed that the photoregulated increase in beta-amylase activity in mustard cotyledon exclusively results from an increase in the rate of de novo synthesis of beta-amylase protein against a very low background rate of enzyme degradation.