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Journal of Biochemistry Advance Access originally published online on March 11, 2009
Journal of Biochemistry 2009 146(1):71-76; doi:10.1093/jb/mvp044
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Sensitive Assay of Glycogen Phosphorylase Activity by Analysing the Chain-Lengthening Action on a Fluologenic Maltooligosaccharide Derivative

Yasushi Makino and Kaoru Omichi*

Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

*To whom correspondence should be addressed. Tel: +81-72-254-9191, E-mail: komichi{at}c.s.osakafu-u.ac.jp

Received February 4, 2009; Accepted March 4, 2009


  Abstract

The action of glycogen phosphorylase (GP) is essentially reversible, although GP is generally classified as a glycogen-degrading enzyme. In this study, we developed a highly sensitive and convenient assay for GP activity by analysing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc4) residue comprising the non-reducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method was used to measure the GP activities of crude and purified GP preparations, and was demonstrated to have about 1,000 times greater sensitivity than the spectrophotometric orthophosphate assay.

Key Words: chain-lengthening action, fluorogenic substrate, glycogen phosphorylase, high-performance liquid chromatography, sensitive assay

Abbreviations: BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Glc, D-glucose; Glc-1-P, glucose-1-phosphate; GlcPA, 1-deoxy-1-[(2-pyridylamino)-D-glucitol]; GP, glycogen phosphorylase; HPLC, high-performance liquid chromatography; PA, pyridylamino; Pi, inorganic phosphate


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