Background and overview[1][2]
The sugar phosphate pathway is an important pathway for sugar metabolism in plants. Its main physiological function is to produce reducing NADPH for biosynthesis, as well as pentose phosphate for nucleic acid metabolism and some intermediate products for the synthesis of amino acids and fatty acids. ; At the same time, it also plays a very important role in maintaining the redox balance of plant cells. Glucose-6-phosphate dehydrogenase catalyzes the irreversible oxidation reaction in the first step of the pentose phosphate pathway and is a key regulatory enzyme. G6PDH in animals is an X-linked inheritance. Its deficiency can lead to red blood cell metabolism disorders, retinal blood vessel occlusion, ketosis, etc. In severe cases, it can be life-threatening. Its overexpression can cause abnormalities in human lipid metabolism, but can increase the lifespan of fruit flies. In plants, many researchers have studied its relationship with plant growth and development and various environmental stresses from various approaches, in order to more clearly explain the pentose phosphate pathway and the possible physiological functions of this enzyme.
Biological properties[1][2]
G6PDH is the rate-limiting enzyme of the pentose phosphate pathway, controlling the carbon flow and reducing power of NADPH in this pathway. The NADPH catalyzed by G6PDH not only provides reducing power for the biosynthesis of certain biological macromolecules in cells, but is also the only reducing power for the regeneration of reduced glutathione (GSH). Therefore, G6PDH plays an important role in the process of cells resisting oxidative stress. plays an important role in.
Preparation[2]
The isolation and purification of glucose-6-phosphate dehydrogenase is as follows: extract the intracellular enzyme from yeast by swelling method in 0.1 mol/L sodium bicarbonate solution. The crude extract is 60% to 75% saturated degree of ammonium sulfate salting out and Sephadex G-100 gel column chromatography. The yield of glucose-6-phosphate dehydrogenase was 12.4%, and the purification factor reached 51.2. The experimental method is:
1) Preparation of crude extract: Take 150g of beer yeast, add 240mL of 0.1mol/L sodium bicarbonate solution, stir at 40℃ for 5h, and centrifuge at 5500r/min for 30min ( 4℃), the supernatant is the crude extract.
2) Purification of enzyme: use ammonium sulfate with saturation of Cabot silica (mass/volume percentage d) of 50%, 60%, 65%, 70%, 75%, 80%, and 85% respectively. The crude extract is precipitated by staged salting out, and the precipitate is collected by centrifugation. Take the salting out precipitate with a saturation of 60% to 75%, dissolve it in 0.05mol/LTris-HCl (pH7.8) buffer, and condense it on SephadexG-100. Gel chromatography column (3cm×36cm), elute with the same buffer (chromatography speed: 0.6mL/min).
3) Determination of enzyme activity: Add 3mL0.05mol/LTris-HCl buffer, 30μL0.05mol/LNADP solution, 20μL1mol/LG-6-P solution into a quartz cuvette, mix well, and then add 10μL enzyme Liquid, quick
After rapid mixing, immediately measure the absorbance change value at 340nm and calculate the enzyme activity. The amount of enzyme that catalyzes the conversion of 1 μmol of substrate into product per minute is defined as one enzyme activity unit (U).
Apply[3]
G6PD is widely present in various cells of the human body and is the rate-limiting enzyme of the pentose phosphate pathway. During this metabolic process, reduced nicotinamide phenylacetine dinucleotide phosphate (NADPH) is produced, which is an important component in all types of cells. The most important reducing agent. Especially in red blood cells, the pentose phosphate pathway is the only source of NADPH, and the defense mechanism against oxidative damage is highly dependent on G6PD activity. Therefore, red blood cells in G6PD-deficient patients are extremely vulnerable to oxidative stress damage.
The body’s antioxidant system mainly includes catalase, superoxide dismutase and glutathione. Although all antioxidant systems are important for cell survival, G6PD has a unique role.
First of all, glutathione and thioredoxin systems have important antioxidant functions in cells. NADPH is an important substrate necessary for these two systems. It converts oxidized glutathione and sulfur oxidized proteins into their reduced forms, thereby exerting antioxidant effects.
Secondly, catalase is another important antioxidant enzyme in cells. It converts hydrogen peroxide into water and oxygen. This process does not require NADPH, but catalase has specific NADPH binding. site, NADPH has an allosteric effect and keeps catalase in an activated state after binding. Although there are other sources of NADPH synthesis in cells, research has found that G6PD is the most important pathway, which is responsible for the normal function of the antioxidant system and cell growth.existence is of great significance. Therefore, reduced G6PD activity and reduced NADPH levels will directly affect the normal function of these antioxidant systems and cause damage to cells and organs.
Main reference materials
[1] Research progress on glucose-6-phosphate dehydrogenase in higher plants
[2] Isolation and purification of glucose-6-phosphate dehydrogenase
[3] The role and mechanism of glucose-6-phosphate dehydrogenase in diabetes and its complications
