Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds

Publisher Website
Google Scholar
Abstract
The biosynthetic mechanism of .alpha.-amylase synthesis in germinating rice (O. sativa L. cv. Kimmaze) seeds was studied in vitro and in vivo. Special attention was focused on the glycosylation of the enzyme molecule. Tunicamycin inhibited glycosylation of .alpha.-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic of glycosylation did not significantly alter enzyme activity. In an in vitro system usig poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of .alpha.-amylase (precursor I) is formed. Inhibition of glycosylation by tunicamycin allowed detection of nonglycosylated precursor (II) of .alpha.-amylase. The MW of the nonglycosylated precursor II produced in the presence of tunicamycin was 2900 daltons less than that of the mature form of .alpha.-amylase (44,000) produced in the absence of tunicamyin and 1800 daltons less than the in vitro synthesized molecule. The inhibition of glycosylation of tunicamycin as well as in vitro translation helped clarify the heterogeneity of .alpha.-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms and fluorography were employed on the separated isozyme components. The mature and tunicamycin-treated nonglycosylated forms of .alpha.-amylase consisted of 3 isozymes. The in vitro translated precursor forms of .alpha.-amylase consisted of 4 multiple components. Apparently, heterogeneity of .alpha.-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice .alpha.-amylase isozymes are encoded by multiple genes.