Sensitive Assay of Glycogen Phosphorylase Activity by Analysing the Chain-Lengthening Action on a Fluologenic Maltooligosaccharide Derivative
| Autor: | Yasushi Makino, Kaoru Omichi |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Swine
Aminopyridines Oligosaccharides Sensitivity and Specificity Biochemistry High-performance liquid chromatography Fluorescence spectroscopy Substrate Specificity Glycogen phosphorylase chemistry.chemical_compound Residue (chemistry) Animals Humans Phosphorylase a Phosphorylase b Phosphorylation Maltose Muscle Skeletal Molecular Biology Chromatography High Pressure Liquid Fluorescent Dyes chemistry.chemical_classification Chromatography Tissue Extracts Glycogen Phosphorylase Glucosephosphates Substrate (chemistry) General Medicine Fluorescence Enzyme Liver chemistry Glycogen Phosphorylase Muscle Form Rabbits Liver Extracts Derivative (chemistry) |
| Zdroj: | Journal of Biochemistry. 146:71-76 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/jb/mvp044 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|