br Materials and methods br

Materials and methods
Results and discussion In our previous studies (Matarneh et al., 2017), mitochondria were mechanically disrupted and separated into supernatant and pellet fractions by centrifugation. The causative agent for enhnced glycolytic flux was shown to be a protein that resides in the supernatant (soluble). In the present study, the β-subunit of mitochondrial F1-ATPase was chosen as a candidate, primarily because it is a water-soluble protein that has the potential to affect elastase through increasing the rate of ATP hydrolysis (Hudson, 2012). For our hypothesis to be correct, the β-subunit must reside in the supernatant fraction. Therefore, western blot analysis was used to confirm the presence of the β-subunit in the supernatants. The difference in β-subunit abundance between total mitochondrial preparation, supernatants, and pellets is readily apparent (see Fig. 1). The supernatant fraction contained >8 times greater (P<0.0001) β-subunit content compared to that of pellet fraction. These data affirmed that the F1-ATPase was dissociated from the mitochondrial inner membrane and re-localized in the soluble fraction. To test the role of mitochondrial F1-ATPase in postmortem metabolism, we utilized our previously described in vitro system that mimics postmortem glycolysis (England et al., 2014, Matarneh et al., 2017). Either 0 or 0.5mg/ml isolated mitochondria were incorporated into the in vitro system in the presence or absence of Na-azide. Na-azide is a potent noncompetitive inhibitor that tightly traps ADP at the catalytic site in the β-subunit of F1-ATPase. This trapping stabilizes the ADP-inhibited state of the enzyme, thereby preventing its ATPase activity (Bowler et al., 2006, Ishii et al., 2014, Vasilyeva et al., 1982). The pH of the in vitro system was significantly affected by the interaction between treatment and time (P<0.0001; Fig. 2). No difference in pH between treatments was detected through 120min. Yet, at 1440min, samples containing only mitochondria had the lowest pH value (P<0.0001), while control and Na-azide samples were the highest. When mitochondria were combined with Na-azide, however, pH decline was attenuated, as evidenced by the elevated pH value at 1440min compared to the mitochondria only treatment. These results are in agreement with our previously published findings where the inclusion of mitochondria to an in vitro model extended pH decline (Matarneh et al., 2017). In the current study, Na-azide was responsible for about 65% inhibition of mitochondria-induced pH decline. These results may seem to contradict our previously published findings (Matarneh et al., 2017), where mitochondria-induced glycolytic flux was maintained in the presence of oligomycin. Oligomycin is an inhibitor that blocks the passage of H+ through the F0 domain, and therefore preventing both ATP synthesis and hydrolysis activiteis of F1F0 ATP synthase (Penefsky, 1985). However, the F1 domain can dissociate and becomes oligomycin insensitive and exhibits a high level of ATPase activity (Dubinsky, 1987, Feinstein and Moudrianakis, 1984), which may explain how mitochondria maintained their effect in the presence of oligomycin. To continue defining the relationship between mitochondrial F1-ATPase and the flux through glycolysis, lactate, glycolytic metabolites, ATP, and IMP were measured in the in vitro system. During postmortem metabolism, pyruvate generated through glycolysis is converted to lactate by lactate dehydrogenase. This reaction is required for a continuous supply of NAD+ to maintain flux through glycolysis under anaerobic conditions. Treatment differentially affected lactate concentration of the in vitro system over time (treatment×time, P=0.0005, Fig. 3). While no difference in lactate was detected at 120min, samples containing mitochondria without Na-azide had the highest lactate level at 1440min (P=0.002) in comparison to the rest of the treatments.