SPECIFIC DETERMINATION OF STARCH HYDROLYZING ENZYME-ACTIVITIES OF LEAVES

Endo- and exoamylase and debranching enzyme were isolated from wheat leaves and used to test the validity of assays proposed to be specific for determining discrete leaf amylolytic activities. Procedures using a blocked PNP-linked G7-maltooligosaccharide substrate and a mixture of PNP-linked GS- and G6maltooligosaccharides were specific for wheat endo- and exoamylase, respectively. An alternative procedure (named «βββ») for specifically assaying endoamylase with β-limit dextrin as the substrate was also developed and confirmed to be specific for wheat leaf endoamylase. These assay procedures were applied to crude leaf extracts from a variety of species. The procedure using PNPGS/G6 as substrate appears to be of widespread value as a sensitive and reliable assay of exoamylase activity in leaf extracts. Endoamylase activity in the extracts was in most casesvery low with BPNPG7 as substrate; the βββ procedure was in general more sensitive in detecting this type of activity. The ratio of the activity units obtained with the βββ procedure to those measured with BPNPG7 as substrate varied considerably with the species investigated. The suitability of using BPNPG7 to assay leaf endoamylase activity may depend on the relative ability of the corresponding enzyme to attack small oligosaccharide and larger branched substrates, respectively. © 1990, Gustav Fischer Verlag, Stuttgart. All rights reserved.