New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme
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ORIGINAL ARTICLE
New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme Miyu Sakaguchi 1 & Yasushi Makino 1
&
Hiroshi Matsubara 1
Received: 5 June 2020 / Revised: 3 October 2020 / Accepted: 15 October 2020 / Published online: 17 November 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1–4Glcα1–4Glcα1–4Glcα1–4(Glcα1–4Glcα1– 4Glcα1–4Glcα1–6)Glcα1–4Glcα1–4Glcα1–4GlcPA (B4/84, where Glc = D -glucose and GlcPA = 1-deoxy-1-[(2pyridyl)amino]-D-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1–4Glcα1–4Glcα1–4Glcα1–4(Glcα1–4Glcα1–4Glcα1–4Glcα1– 6)Glcα1–4Glcα1–4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1– 6Glcα1–4Glcα1–4GlcPA (B3/31) was prepared from commercially available Glcα1–6Glcα1–4Glcα1–4Glc. Using αcyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1–4Glcα1–4Glcα1–4Glcα1– 4(Glcα1–6)Glcα1–4Glcα1–4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase. Keywords Fluorogenicbrancheddextrin . Glycogendebranching enzyme . Glycogen storage disease typeIII . Phosphorylaselimit dextrin . Pyridylamination
Introduction Animal tissues store glycogen, a highly branched macromolecule consisting of thousands of D-glucose (Glc) residues [1, 2]. In the glycogen molecule, most of the Glc residues are connected by α-1,4-glycosidic bonds, while side branches at approximately every tenth Glc residue are created by α-1,6-glycosidic bonds. The full glycogen molecule was reported to have a Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10719-020-09955-7) contains supplementary material, which is available to authorized users. * Yasushi Makino [email protected] 1
Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Gakuen-cho 1-1, Naka-ku, Sakai Osaka, 599-8531 Osaka, Japan
spherical shape with 12 concentric tiers [3]. In response to energy demands, glycogen phosphorylase (GP; EC 2.4.1.1) degrades glycogen in coopera
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