Mak193

Citrate synthase activity in mitochondria was measured with a kit purchased from Sigma-Aldrich (MAK193; St. Louis, MO, USA). The activities of acetyl-CoA and propionyl-CoA carboxylases were measured following the method described by Hugler et al. [40 (link)] with a slight modification. Briefly, homogenates from frozen tissues (0.5–0.65 mg protein) were incubated with acetyl-CoA or propionyl-CoA (0.4 mM) and [¹⁴C]Na₂CO₃ (37 kBq/µmol) in a buffer containing 100 mM Tris/HCl, pH 7.8, 5 mM MgCl₂, 5 mM dithioerythritol, 4 mM ATP, 2 mM NADPH, and 10 mM NaHCO₃ at 37 °C for 6 min. The reaction was started by adding the substrate (acetyl-CoA or propionyl-CoA) and ended by adding HCl. The unused substrate then was removed from the reactant using an analytical evaporator (MULTVAP, Berlin, MA, USA) under N₂.

Mitochondria were isolated from Wt and Akap1^−/− lungs using the mitochondria isolation kit as per the manufacturer’s instructions (Thermo Scientific). Mitochondrial pellets were lysed in 2% CHAPS in TBS supplemented with protease and phosphatase inhibitors, quantified by Pierce BCA protein assay kit (Thermo Scientific) and stored at −80°C. The fumarase activity colorimetric assay kit MAK206 (Sigma-Aldrich, St. Louis, MO), citrate synthase activity kit MAK193 (Sigma-Aldrich) and aconitase activity assay kit MAK051 (Sigma-Aldrich) were used to detect enzymatic activity in Wt and Akap1^−/− mitochondrial fraction (25 μg) according to the manufacturer’s instructions. Aconitase activity (nmole/min/ml), citrate synthase activity (milliunit/mL) and fumarase activity (milliunit/mL) was normalized to the amount of protein used in the assay. Mitochondrial Complex I activity was determined using 25 μg of mitochondrial lysate and Complex I enzyme activity kit (#ab109721, Abcam, Waltham, MA) as per manufacturer’s instructions. The Complex I activity (mOD/min) was normalized to the amount of protein used in the assay.

According to the manufacturer's protocol, citrate synthase (CS) activity was measured using a kit (Catalog no. MAK193, Sigma, St. Louis, MO). Briefly, heart tissue samples were used for each enzymatic analysis. Measurements were performed in triplicate for each experimental condition) at room temperature in 96 well dishes. The enzyme was immunocaptured within the microplate wells, and activity was determined by measuring the color development of TNB, which is generated in the reaction of citrate synthesis. Enzyme activity was measured spectrophotometrically (at an absorbance wavelength of 412 nm). The formula provided by the manufacturer was used to calculate total CS activity.

Citrate synthase (CS) activity was assessed using a commercial colorimetric kit (Sigma-Aldrich, Burlington, MA, USA, MAK193). Kidney tissue was cryo-grinded in a mortar placed in liquid nitrogen, diluted in the provided assay buffer, and thereafter, the manufacturer’s protocol was adhered to. CS activity was normalized to protein content and reported as U/g protein.

NRCM cells were seeded at a density of 10⁶ cells/well in 6-well plates and cultured. After the appropriate treatment, cells were harvested; the cell pellet was suspended in ice-cold citrate synthase cell lysis buffer and, then, centrifuged for 5 min at 4°C at 10,000 x g; then, the supernatant was collected for further use. Citrate synthase was measured using a kit from Sigma Aldrich (MAK193) following the manufacturer's instruction. The absorbance was recorded at 412 nm every 5 minutes for 50 minutes. The colorimetric product (GSH) was proportional to the enzymatic activity of citrate synthase and normalized to the quantity of cells.

Citrate synthase, succinyl-CoA synthetase, and α-ketoglutarate dehydrogenase enzyme activities were measured using the assay kits according to the manufacturer’s protocols (Sigma-Aldrich; MAK193, MAK217, and MAK189). Briefly, each parental strain was cultured in Trypticase soy broth medium for 48 h. The bacterial cell pellets were collected and lysed using the specific assay buffer provided in each kit. The amount of total protein in each strain was measured using the bicinchoninic acid (BCA) protein assay (Pierce/Thermo Scientific) and used as a reference to compare enzyme activities between samples. One-way ANOVA with Tukey’s test for multiple comparisons was used to examine differences in enzyme activities between ground control and space-surviving strains.
The ATP production in each strain was measured by using CellTiter-Glo luminescent cell viability assay according to the manufacturer’s protocol (Promega; G7571). Briefly, each strain was cultured and lysed as described previously above. Lysates with 10 μg total protein per sample were used to compare each sample-specific amount of ATP. One-way ANOVA with Tukey’s test for multiple comparisons was used to analyze the statistical significance of ATP production between ground controls and space-surviving strains.