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J Biol Chem. 2016 Apr 1;291(14):7409-17. doi: 10.1074/jbc.M115.711663. Epub 2016 Jan 28.

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.

Author information

1
From the Department of Biochemistry, the Fraternal Order of the Eagles Diabetes Research Center, the Abboud Cardiovascular Research Center, and the Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
2
From the Department of Biochemistry, the Fraternal Order of the Eagles Diabetes Research Center, the Abboud Cardiovascular Research Center, and the Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242 eric-taylor@uiowa.edu.

Abstract

The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 μm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 μmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

KEYWORDS:

kinetics; metabolism; method; mitochondria; mitochondrial pyruvate carrier; pyruvate; pyruvate transport; transport; uptake

PMID:
26823462
PMCID:
PMC4817172
DOI:
10.1074/jbc.M115.711663
[Indexed for MEDLINE]
Free PMC Article

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