Abstract
The enzymatic function of GAPDH (EC.1.2.1.12) is an integral component of glycolysis in glucose metabolism. Thus, GAPDH’s catalytic capacity profoundly influences the “bioenergetic signature” of any metabolically active cell. Determination of the enzymatic function of GAPDH has been instrumental in the assessment of the glycolytic or overall metabolic capacity of proliferating cells. In this chapter, we describe two different approaches to determine the activity of the GAPDH enzyme, based on quantitative and qualitative analytical methods. The quantitative approach is based on the spectrophotometric principle which relies on the net abundance of NADH, a coenzyme that is either generated or depleted depending upon the direction of the reversible reaction of GAPDH. The method outlined here is based on the reversible reaction, where a decrease in NADH is monitored at the absorption maximum (~340 nm). The advantage of this NADH-based assay is that it is specific to GAPDH. The qualitative approach uses an in-gel activity assay, in which the enzyme-dependent release of pyrophosphate is visualized as a calcium-phosphate precipitate. The qualitative assay is also relevant for kinetic analysis as the amount of calcium-phosphate precipitate relies on the level of GAPDH activity. Both these approaches are invaluable and rely on scientifically sound analytical principles.
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References
Bergmeyer HU, Bergmeyer J, Grassl M (1983, 1986) Methods of enzymatic analysis, 3rd ed. Verlag Chemie, Weinheim, 1986
Ganapathy-Kanniappan S, Kunjithapatham R, Torbenson MS et al (2012) Human hepatocellular carcinoma in a mouse model: assessment of tumor response to percutaneous ablation by using glyceraldehyde-3-phosphate dehydrogenase antagonists. Radiology 262(3):834–845
Hwang NR, Yim SH, Kim YM et al (2009) Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions. Biochem J 423(2):253–264
Kunjithapatham R, Geschwind JF, Devine L et al (2015) Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum. J Proteome Res 14(4):1645–1656
Mounaji K, Erraiss NE, Iddar A et al (2002) Glyceraldehyde-3-phosphate dehydrogenase from the newt pleurodeles waltl. protein purification and characterization of a GapC gene. Comp Biochem Physiol B Biochem Mol Biol 131(3):411–421
Nakajima H, Amano W, Kubo T et al (2009) Glyceraldehyde-3-phosphate dehydrogenase aggregate formation participates in oxidative stress-induced cell death. J Biol Chem 284(49):34331–34341
Rothe G (1994) Electrophoresis of enzymes: laboratory methods. Springer-Verlag, Berlin
Seidler NW (2013) Basic biology of GAPDH. Adv Exp Med Biol 985:1–36
Shibuya A, Ikewaki N (2002) High serum glyceraldehyde-3-phosphate dehydrogenase levels in patients with liver cirrhosis. Hepatol Res 22(3):174–179
Thomas AP, Halestrap AP (1981) Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide. Biochem J 196(2):471–479
Vandooren J, Geurts N, Martens E et al (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10(3):211–220
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Ganapathy-Kanniappan, S. (2017). Analysis of GAPDH Enzyme Activity: A Quantitative and Qualitative Approach. In: Advances in GAPDH Protein Analysis: A Functional and Biochemical Approach. Springer, Singapore. https://doi.org/10.1007/978-981-10-7342-7_2
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DOI: https://doi.org/10.1007/978-981-10-7342-7_2
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