Journal Article

An Assay Method for Glycogen Debranching Enzyme Using New Fluorogenic Substrates and Its Application to Detection of the Enzyme in Mouse Brain

Kaoru Omichi and Sumihiro Hase

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 123, issue 5, pages 932-936
Published in print May 1998 | ISSN: 0021-924X
Published online May 1998 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022027
An Assay Method for Glycogen Debranching Enzyme Using New Fluorogenic Substrates and Its Application to Detection of the Enzyme in Mouse Brain

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An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-α-D-glucosyl-α-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 Glcαl-4(Glcαl-6)Glcαl-4Glcαl-4Glcαl-4GlcPA (FD6) and Glcαl-4Glcαl-4(Glcαl-6)Glcαl-4Glcαl-4Glcαl-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.

Keywords: assay methods; fluorogenic substrate; glycogen debranching enzyme; HPLC.

Journal Article.  0 words. 

Subjects: Biochemistry

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