title: Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin.
abstract: Glycogen phosphorylase (GP) exists in two interconvertible forms, GPa (phosphorylated form, high activity) and GPb (nonphosphorylated form, low activity). Phosphorylase kinase (PhK) catalyses the phosphorylation of GPb and plays a key role in the cascade system for regulating glycogen metabolism. In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (ΔA) was calculated by the following formula: ΔA = A(+) - A(0), where A(+) and A(0) represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 µg of GPb per sample was needed to obtain A(+) and A(0) values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity.
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Miyagawa
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8531, Japan ymakino@c.s.osakafu-u.ac.jp.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Makino
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sato
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003912:Dextrins / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006003:Glycogen / Q000378:metabolism*; D024982:Glycogen Phosphorylase, Muscle Form / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D003912:Dextrins; D009844:Oligosaccharides; C016549:maltohexaose; C013862:caloreen; D006003:Glycogen; D024982:Glycogen Phosphorylase, Muscle Form; D010764:Phosphorylase Kinase
keywords: fluorogenic dextrin; glycogen; glycogen phosphorylase; nonradioactive assay; phosphorylase kinase