An assay method for glycogen debranching enzyme using new fluorogenic substrates and its application to detection of the enzyme in mouse brain

An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed, Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives, Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glc alpha 1-4(Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4-Glc alpha 1-4Glc-PA (FD6) and Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc-Pa (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.