DEMONSTRATION OF IN-VITRO STARCH BRANCHING ENZYME-ACTIVITY FOR A 51 50-KDA POLYPEPTIDE ISOLATED FROM DEVELOPING BARLEY (HORDEUM-VULGARE) CARYOPSES/

Starch branching enzyme (SBE, EC 2.4.1.18) activity was followed in de veloping barley (Hordeum vulgare L. cv. Golf) caryopses during a perio d of one month after anthesis. Caryopses with the highest specific act ivity, and corresponding to a fresh weight of around 60 mg per caryops is, were homogenized and the soluble extract used for branching enzyme purification by FPLC chromatography. Four branching enzyme activity f ractions were resolved. From one of these fractions, which exhibited h igh activity in both the phosphorylation stimulation and amylose branc hing assays, a branching enzyme preparation containing two related pol ypeptides of 51 and 50 kDa was obtained. Native polyacrylamide gel ele ctrophoresis and gel filtration showed that the 51/50-kDa polypeptide is monomeric. A combination of phosphorylation stimulation and amylose branching gel assays, SDS-PAGE, and TLC was used to demonstrate the b ranching activity of the 51/50-kDa polypeptide. The activity was furth er confirmed by spectroscopic analyses of iodine-glucan complexes. SBE s from four different plant species were compared using the phosphoryl ation stimulation gel assay.