Glycolysis related experiments.pptx
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Experiments related to glycolysis include measuring extracellular glucose and lactate levels, the extracellular acidification rate, and the activity of rate-limiting glycolytic enzymes. Extracellular glucose and lactate are measured using kits to determine levels in cell cultures. The extracellular acidification rate measures pH changes from lactic acid excretion over time as an indicator of glycolysis. Rate-limiting enzymes like hexokinase, phosphofructokinase, and pyruvate kinase can be assayed by coupling them with NAD+/NADH or NADP+/NADPH dependent reactions and measuring absorbance changes at 340nm.
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1. Carbohydrates are digested into monosaccharides like glucose in the small intestine through the actions of digestive enzymes.
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Glycolysis.pptx
Glycolysis is the process by which glucose is broken down to pyruvate through a series of enzymatic reactions to generate ATP. It occurs in two phases: the preparatory phase where glucose is phosphorylated and the payoff phase where energy is generated. Glycolysis is regulated by hormones and key enzymes. The fate of pyruvate includes conversion to lactate or acetyl-CoA to feed into the TCA cycle. Gluconeogenesis and the pentose phosphate pathway are important glucose production and antioxidant pathways respectively.
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Glycolysis related experiments.pptx
- 3. Glucose
- 4. Glycolysis?
- 6. AnaerobicGlycolysis?!! Anaerobic glycolysis is the transformation of glucose to lactate when limited amounts of oxygen (O2) are available WHEN???
- 7. Glycolysis related experiments Measurement of extracellular glucose and lactate Extracellular acidification rate Measuring the activity of rate-limiting glycolytic enzymes
- 8. Measurement of extracellular glucose and lactate Glucose and lactate are measured in extracellular fluid obtained from cell cultures by glucose kits spectrophotometrically
- 9. Extracellular acidification rate Glycolysis is determined through measurements of the extracellular acidification rate (ECAR) of the surrounding media, which is predominately from the excretion of lactic acid per unit time after its conversion from pyruvate. ECAR is essentially a measurement of pH, a buffering agent, such as sodium bicarbonate.
- 10. Measuring the activity of rate-limiting glycolytic enzymes
- 12. Hexokinase, phosphofructokinase, and PK are the main rate-limiting enzymes in glycolysis. Conversion from NAD+ to NADH or NADP+ to NADPH is often used in enzymatic assays because NADH and NADPH absorb light at 340 nm, while NAD+ and NADP+ lack absorbance at this wavelength. By coupling the enzyme of interest with an NAD+/NADH- or NADP+/NADPH dependent enzyme, the activity of the enzyme can be measured by the change in absorbance at 340 nm over time. Hexokinase, phosphofructokinase, and PK can be coupled with NAD+/NADH- or NADP+/NADPH-dependent enzymes by using enzymes that react with their products e.g. coupling glucose-6-phosphate dehydrogenase in hexokinase assay