Sensitive assay of glycogen phosphorylase activity by analysing the chain-lengthening action on a Fluorogenic [corrected] maltooligosaccharide derivative.
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 (Glc(4)) 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.
Makino
Yasushi
Y
Department of Chemistry, Osaka Prefecture University, Sakai, Japan.
Omichi
Kaoru
K
D000631:Aminopyridines; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D024981:Glycogen Phosphorylase / Q000097:blood / Q000378:metabolism*; D024982:Glycogen Phosphorylase, Muscle Form / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D008110:Liver Extracts / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives*; D018482:Muscle, Skeletal / Q000201:enzymology; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D010762:Phosphorylase a / Q000378:metabolism; D010763:Phosphorylase b / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013552:Swine; D014020:Tissue Extracts / Q000378:metabolism
D000631:Aminopyridines; D005456:Fluorescent Dyes; D005958:Glucosephosphates; D008110:Liver Extracts; D009844:Oligosaccharides; D014020:Tissue Extracts; D008320:Maltose; C031590:glucose-1-phosphate; D024981:Glycogen Phosphorylase; D024982:Glycogen Phosphorylase, Muscle Form; D010762:Phosphorylase a; D010763:Phosphorylase b; C032439:alpha-aminopyridine