Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics |
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Affiliation: | 1. Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, People''s Republic of China;2. State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, People''s Repulic of China;1. Instituto de Ciências Biológicas (ICB), Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil;2. Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil;3. Instituto de Oceanografia (IO), Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil;4. Programa de Pós-Graduação em Aquicultura, Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil |
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Abstract: | The enzyme glucose-6-phosphatase (G-6-Pase) catalyzes the hydrolysis of glucose-6-phosphate (G-6-P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G-6-Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three-part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G-6-Pase. The assessment of G-6-Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (Pi) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G-6-Pase with potential clinical importance. The format of the three-part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease. |
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