Journal
ANALYTICAL BIOCHEMISTRY
Volume 340, Issue 2, Pages 279-286Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.02.016
Keywords
glycogen debranching enzyme; fluorogenic substrate; branched dextrin; enzyme assay; HPLC
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Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4GlcPA (B3), Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (B4), Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (B5), Glc alpha 1-4Glc alpha 1-4(Gl alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Gle alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (B6), Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6) Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (B7), and Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product Was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose. and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay. (c) 2005 Elsevier Inc. All rights reserved.
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