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Metabolism and Enzymology

-Amylase from Mustard (Sinapis alba L.) Cotyledons ¹

Immunochemical Evidence for Synthesis de Novo during Photoregulated Seedling Development

School of Life Sciences, University of Hyderabad, Hyderabad—500 134, India

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

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