Kit and method for measuring activity of GCS (glycogen synthetase)

Abstract

The invention discloses a kit and a method for measuring activity of GCS (glycogen synthetase). The kit comprises reagents as follows: an extraction solution comprising a Tris-HCl buffer solution, a reagent I prepared by dissolving Tris, magnesium chloride, potassium chloride, potassium hydroxide and hydrochloric acid in distilled water, a reagent II prepared by dissolving Tris, magnesium chloride, EDTA (ethylene diamine tetraacetic acid), mercaptoethanol and hydrochloric acid in distilled water, a reagent III comprising lactic dehydrogenase, a reagent IV comprising phosphoenolpyruvic acid, NADH (nicotinamide adenine dinucleotide) and pyruvate kinase as well as a reagent V comprising glycogen, UDPG (uridine diphosphate glucose) and glucose-6-phosphate. According to the method, under the combined action of pyruvate dehydrogenase and the lactic dehydrogenase, the oxidation rate, measured at 340 nm by a spectrophotometer, of NADH reflects the production rate of a reaction product UTP (uridine triphosphate) of GCS, the activity of GCS is further reflected, and the method is high in sensitivity. Currently, no effective kit for measuring the activity of the GCS under the combined action of enzymes on the basis of the spectrophotometry is commercially available.

Description

technical field [0001] The invention belongs to the field of life sciences and relates to a kit, in particular to a glycogen synthase activity assay kit and a method thereof. Background technique [0002] Glycogen synthase (GCS) catalyzes UDPG and glucose residues to generate glycogen and UTP, and connects and extends sugar chains with α-1,4-glycosidic bonds. It is the rate-limiting enzyme of glycogen synthase in liver and muscle, and is the action of insulin The main target enzyme plays an important role in the regulation of glucose metabolism and the maintenance of blood glucose homeostasis. [0003] Currently available methods for detecting glycogen synthase in the market are rinsing method and solid-phase filter paper method. Both methods are based on UDP 14 Under the action of GS, CG synthesizes radioactive glycogen, and uses rinsing or solid phase filter paper to separate radioactively labeled glycogen and free UDP 14 CG, Finally, a method for measuring radiation in...

AI Technical Summary

Problems solved by technology

The rinsing method requires a large amount of 70% absolute ethanol for rinsing, which takes a long time and is likely to cause environmental pollution

Although the solid-phase filter paper method overcomes the disadvantages of environmental pollution, there are still several disadvantages as follows: 1. The extract of glycogen synthase has too many components and the cost is high; 2. This method needs to use radiolabeled UDP 14 CG, the reagent cost is high; 3. The separation process is cumbersome and time-consuming; 4. The equipment used-liquid scintillation counter is not commonly used in general laboratories