A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity

Lawrence R Gray; Adam J Rauckhorst; Eric B Taylor

doi:10.1074/jbc.M115.711663

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A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity

Journal article

Open access

Peer reviewed

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity

Lawrence R Gray, Adam J Rauckhorst and Eric B Taylor

The Journal of biological chemistry, Vol.291(14), pp.7409-7417

04/01/2016

DOI: 10.1074/jbc.M115.711663

PMCID: PMC4817172

PMID: 26823462

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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.

Mitochondrial Membrane Transport Proteins - metabolism

Pyruvic Acid - chemistry

Animals

Anion Transport Proteins - analysis

Mitochondria, Liver - metabolism

Mitochondrial Membrane Transport Proteins - analysis

Pyruvic Acid - metabolism

Rats

Mice

Anion Transport Proteins - metabolism

Rats, Sprague-Dawley

Details

Title: Subtitle: A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity
Creators: Lawrence R Gray - 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
Adam J Rauckhorst - 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 B Taylor - 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
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.291(14), pp.7409-7417
DOI: 10.1074/jbc.M115.711663
PMID: 26823462
PMCID: PMC4817172
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: F32 DK101183 / NIDDK NIH HHS R01 DK104998 / NIDDK NIH HHS T32 HL007121 / NHLBI NIH HHS T32 HL007638 / NHLBI NIH HHS R01DK104998 / NIDDK NIH HHS R00 AR059190 / NIAMS NIH HHS P30CA086862 / NCI NIH HHS P30 CA086862 / NCI NIH HHS
Language: English
Date published: 04/01/2016
Academic Unit: Molecular Physiology and Biophysics
Record Identifier: 9984025306602771

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