Determination of key enzymes and important products in the anaerobic oxidation pathway

Summary

Glucose undergoes a 10-step metabolic reaction of glycolysis to produce keto acid, which is then further produced as lactate or decarboxylated into acetyl coenzyme A (CoA) in the mitochondria to enter the citric acid cycle. In the metabolic pathway of anaerobic oxidation of glucose to lactate, there are four key enzymes that determine the speed and direction of the reaction, namely hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PFK), and pyruvate kinase (PFK). , pyruvate kinase (PK) and lactic dehydrogenase (LDH). Important products are pyruvate and lactate. The key enzymes and important products in the anaerobic oxidation pathway assay experiment is the key enzymes and products produced in the metabolic pathway of anaerobic oxidation of glucose to lactate. Currently, there are 6 methods for the determination of key enzymes and important products in the anaerobic oxidation pathway: HK1 activity assay, PFK-1 activity assay, PK activity assay, LDH activity assay, pyruvate content assay and lactate content assay.

Principle

The basic principle of the determination of key enzymes and important products in the anaerobic oxidation pathway is that the key enzymes and products produced in the metabolic pathway of anaerobic oxidation of glucose to lactic acid can be detected by colorimetric analysis using a spectrophotometer or an enzyme marker.

Operation method

HK1 Activity assay

Principle

The basic principle of the HK1 activity assay is that glucose and ATP are catalyzed by HK to generate glucose-6-phosphate and ADP; glucose-6-phosphate dehydrogenase (G6PDH)-catalyzed and oxidized nicotinamide adenine dinucleotide phosphate (NADP) is present to generate glucose-6-phosphate and reduced NADP (NADPH). In the presence of oxidized nicotinamide adenine dinucleotide phosphate (NADP) and catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate lactone and reduced NADP are produced. In the presence of sufficient NADP, the change in NADPH content is proportional to the change in glucose-6-phosphate content. Since NADPH has a maximum absorption peak at 340 nm, the change in absorbance of NADPH can reflect the activity of HK.

Materials and Instruments

1、Cell samples or tissue samples
2、Cell lysate
3、Saline

Move

The basic procedure of HK1 activity assay can be divided into the following steps:

① Cell samples: collect cells into a centrifuge tube, discard the supernatant, add 200 μl of cell lysate to every 10° cells, homogenize thoroughly, centrifuge at 8000 g for 10 minutes, remove the supernatant, and place on ice for measurement;

② Tissue samples: add 100 μl of physiological saline to every 10 mg of tissue, homogenize, centrifuge at 4 ℃ and 8000 g for 10 minutes, remove the supernatant and place on ice for testing;

③ Serum (plasma) samples can be tested directly. All samples should avoid repeated freezing and thawing which may affect the enzyme activity.

Caveat

① The HK activity of different cells and tissues varies greatly, and it is necessary to carry out pre-tests to determine the measurement parameters and the degree of sample dilution;② The analysis of NADPH is greatly affected by temperature, and the temperature of the reaction solution must be maintained at a constant temperature of 37℃;③ Some reagents need to be stored at low temperature to avoid denaturation and inactivation.

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