Abstract
Pyruvate dehydrogenase complex (PDC) and pyruvate carboxylase (PC) are mitochondrial enzymes that provide the initial steps of the two main alternatives for pyruvate metabolism: oxidative decarboxylation vs. anaplerotic carboxylation, gluconeogenesis, and glycerogenesis. Assays of the enzymatic activity of these two enzymes in cells and tissues are described in this chapter, based on evolution or fixation of 14CO2. These assays are both suitable for use in crude homogenates of cultured skin fibroblasts, lymphocytes, and frozen muscle (PDC) or liver (PC). Activities of these two enzymes are related to spectrophotometric assays of two other mitochondrial enzymes, dihydrolipoamide dehydrogenase (E3) and citrate synthase (CS), providing initial indices of sample integrity and mitochondrial content. These parameters have proven useful for initial detection of inherited human disorders due to deficiencies of these enzymes, and in combination with available genetic analyses can lead to confirmation of specific diagnoses.
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Acknowledgments
The authors are indebted to the late Merton Utter and his associates, whose biochemistry laboratory at Case Western Reserve University was responsible for initial development of these assays; to Mulchand Patel and his associates who continued this work; and to Marilyn Lusk, who trained many members of the CIDEM staff to perform and refine these methods for their clinical application.
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Kerr, D., Grahame, G., Nakouzi, G. (2012). Assays of Pyruvate Dehydrogenase Complex and Pyruvate Carboxylase Activity. In: Wong, Ph.D., LJ. (eds) Mitochondrial Disorders. Methods in Molecular Biology, vol 837. Humana Press. https://doi.org/10.1007/978-1-61779-504-6_7
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DOI: https://doi.org/10.1007/978-1-61779-504-6_7
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