Assessment of Mitochondrial Protein Glutathionylation as Signaling for CO Pathway

Abstract

Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.

Brain Glucose Metabolism: Integration of Energetics with Function

“…Interestingly, 14 CO 2 production from [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose and [6-14 C]glucose did not change with serotonin treatment, whereas decarboxylation of [1-14 C]glucose rose considerably. Presumably, the net rise in shunt flux determined with [1-versus 6-14 C]glucose also involves [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose with its label scrambling via the shunt. Ignoring Fru-6-P recycling, the rise in glycolysis during serotonin treatment might be roughly estimated by dividing the net rise in shunt flux rate in Table II 3 H can also be transferred to other compounds, such as fatty acids and glycerol in rat epididymal fat pad slices (337), reducing the amount in water.…”

“…An in vivo approach is to inhibit 6-phosphogluconate dehydrogenase and measure the rate of incorporation of 14 C into 6-phosphogluconate from [U-14 C]glucose over short time intervals (205). More recent MRS assays in vitro and in vivo include use of [2-or 3- 13 C]glucose (67), [1,[2][3][4][5][6][7][8][9][10][11][12][13] C 2 ]glucose (66,183), and [1,[6][7][8][9][10][11][12][13] 532) provided evidence for recycling of Fru-6-P back to Glc-6-P via PGI and re-entry into the shunt (FIGURE 1). They showed that when pentose shunt activity measured as 14 CO 2 production from ( 14 C 1 -14 C 6 )glucose and was reduced by 50% with dehydroepiandrosterone (DHEA) to inhibit 6-phosphogluconate dehydrogenase, there was a twofold increase in 3 H 2 O release from [3-3 H]glucose (ascribed to glycolysis, but see below) and a 3.5-fold rise in [Glc-6-P].…”

“…This approach is also subject to uncertainties because of label scrambling. Baquer and colleagues (35) assayed pentose shunt activities based on 14 CO 2 produced from [1versus 6-14 C]glucose relative to glycolytic plus PDH rate that was determined by 14 CO 2 production from [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose (via decarboxylation of the generated [1- 14 C]pyruvate by PDH). The relative rates in brain slices from 1-dayold and adult rats were quite low,~9 and 1%, respectively.…”

“…Use of [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose assumes decarboxylation of two molecules of [1- 14 C]pyruvate per [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose at the PDH step, with no loss of labeled lactate. However, [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose would also enter the pentose shunt (just as [1-14 C and 6-14 C]glucose), and label scrambling would occur. From three Glc-6-P entering the shunt, five pyruvate are produced, three labeled in carbon 1 that are lost at the PDH step, one labeled in carbons 1 and 2, and one labeled in carbon 2, and as shown by Gaitonde et al (206) C]glucose with its release to the medium would increase the magnitude of underestimation of glycolysis/oxidation, and true pentose shunt rates relative to glycolysis would be even lower than reported.…”

“…Oxidation of glucose-derived lactate during acoustic stimulation of awake rats was shown to be small. This was directly tested by microinfusion of [3,[4][5][6][7][8][9][10][11][12][13][14] C]glucose into the inferior colliculus and analysis of [ 14 C]lactate and 14 CO 2 in the microdialysate. 14 CO 2 is released at the PDH step, so if lactate is produced, released to interstitial fluid, quickly taken up by another cell, and oxidized, then 14 CO 2 and [ 14 C]lactate concentrations in the microdialysate would be similar (disregarding any label scrambling via the pentose shunt discussed previously).…”