An Endogenous α-Amylase Inhibitor in Barley Kernels

Abstract

Barley (Hordeum distichum cv Klages) kernels were shown to contain a factor that converted malted barley a-amylase II to the a-amylase III form. After purification by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gelfiltration on Bio Gel P60, the factor gave a single band of protein on isoelectric focusing. The purified factor inhibited hydrolysis of soluble starch by a-amylase II from malted barley and germinated wheat (Triticum aestivum cv Neepawa). However, a-amylase I from these cereals was not affected. The inhibitor was not dialyzable and was retained by a PM 10 ultraffitration membrane suggesting a molecular weight greater than 10,000 daltons. Heat treatment of the inhibitor at 70°C for 15 minutes at pH 5.5 and 8.0 resulted in considerable loss of inhibitory activity. Isoelectric focusing studies on crude extracts of germinated barley kernels have shown previously that the total a-amylase activity was distributed among three groups of enzyme bands (8). The groups were designated a-amylases I, II, and III, in order of increasing isoelectric point. Quantitative extraction of enzymes from focused gels showed that a high proportion of total activity was found in the a-amylase III group (8). However, when the applied sample was pretreated for 15 min at 70°C, there was a significant decrease in a-amylase III activity and a corresponding increase in a-amylase II activity. This suggests that the heat treatment converted a-amylase III to a-amylase II. Furthermore, a-amylases II and III were shown to share immunochemical identity (9). Preliminary investigation revealed that kernels of mature barley contained a factor that appeared to convert a-amylase 11 to a-amylase III and also to inhibit a-amylase II. Protein-like inhibitors of native a-amylases have been reported previously in winter wheat (15, 16) and in one cultivar of maize (1). Activation, after affmity chromatography, ofa-amylase in extracts from germinated triticale kernels suggested that triticale also may contain enzyme inhibitors (17). The present investigation was undertaken to purify and characterize the factor in mature barley responsible for interconversion of malted barley a-amylases II and III and for inhibition of a-amylase II.