Oscillations of NADH and ATP caused by gradual entry of pyruvate within mitochondria
First of all, we investigated the NADH oscillation when pyruvate entered within mitochondria. When pyruvate passes across mitochondrial inner membrane, mitochondrial pyruvate carrier (MPC), which is protein embedded within membrane, facilitates the pass of pyruvate across membrane 14–15. After a reaction of pyruvate with NAD+ and CoA within inner membrane of mitochondria, reactions of citric acid cycle and respiratory chain proceed. In order to ascertain the existence of NADH oscillation in these processes, at first, isolated mitochondria were dispersed in the Tris-buffer solution (2mL) containing sodium pyruvate in a cuvette and the time course of the fluorescence of NADH was measured at 340 nm (exciting light) and 460 nm (emission light). Figure 1a shows that the concentration of NADH decreased with oscillation at the condition of 50mM pyruvate. Further decreasing in pyruvate concentration led to disappearance of oscillation gradually.
Then, ADP, which was the substrate of ATP synthase, was also added to the solution of mitochondria and pyruvate. The time course of fluorescence intensity due to NADH is shown in Fig. 1b. NADH oscillation occurred in the same way even when ADP was added.
Further, in order to verify the generation and oscillation of ATP, 1mM luciferin (0.1mL), 1mg/mL luciferase (0.1mL) and 1mM Mg2+ ion (0.05mL) was added to the solution of mitochondria and pyruvate. Fluorescence was observed at the wave lengths of 550nm. The oscillation of ATP, however, was not observed at the same condition as those of Fig. 1a and 1b.
As shown in Fig. 1c, short-period oscillation of ATP (10min ~ 20min) was observed in the concentration range of 20 mM or less of pyruvate at fixed concentration of ADP (6.67mM) after induction period. The phase diagram, which demonstrates the range of concentrations of pyruvate and ADP where oscillatory reaction happened, is shown in Fig. 1d.
In order to ascertain that florescence by NADH and ATP was due to which inside or outside of mitochondria, fluorescence was measured after removing mitochondria by centrifugation. The result showed that fluorescence was due to outer solution of mitochondria. NADH and ATP produced in mitochondria should flow out or flow in corresponding to the concentration difference between inside and outside of mitochondria membrane. Consequently, observed oscillations strongly support that the oscillations take place in mitochondria.
Oscillations of NADH and ATP in the process via malate-aspartate shuttle
NADH produced in glycolysis is also utilized in the respiratory chain. Although NADH is not possible to permeate through membrane, NAD+ is possible to permeate through membrane and is converted to NADH by utilizing malate-aspartate shuttle 13. So, we investigated the oscillation in the case of adding mitochondria to the solution containing NAD+ and malate by measuring the fluorescence. The fluorescence of NADH was measured at 340 nm (exciting light) and 460 nm (emission light).
Figure 2a shows that NADH oscillation occurred with a decrease in NADH concentration in a wide range of NAD+ and malate in the absence of ADP supply. When ADP was added in this system, as shown in Fig. 2b and 2c, oscillations of both NADH and ATP with short period and small amplitude happened, being different from that of pyruvate solution. Furthermore, the oscillation lasted for long time and occurred in higher concentration of ADP compared to that caused by gradual entry of pyruvate. It was found that oscillation occurs without going through the process of citric acid cycle.
Then, we investigated the time course of pH within mitochondria for both systems of permeations of pyruvate and NAD+ by using pH-dependent fluorescent reagent. The results are shown in Fig. 2d and 2e. In both systems, pH oscillation was also observed as well as those of NADH and ATP.
In addition to the investigation with mitochondria, in order to verify an assumption that NADH and the other intermediates oscillations triggered by the gradual entry of first substrates (pyruvate or NADH) occur continuously in the citric acid cycle and the respiratory chain, we carried out the following experiments by using dialysis membrane.
Oscillations of respiratory chain intermediates using semipermeable membrane as a model of mitochondria membrane
In addition to NADH produced by the citric acid cycle, NADH produced by glycolysis is also utilized in the respiratory chain. Although NADH is not able to permeate through mitochondrial membrane, NADH is produced from NAD+ within matrix of mitochondria by malate-aspartate shuttle 13. In order to prove an assumption that the gradual entry of NADH caused oscillatory reactions of intermediates existing in respiratory chain, a method using dialysis membrane was employed as a simple model of permeation through membrane.
At first, the direct reaction between NADH and coenzyme Q (CoQ) was investigated. NADH solution was put in upper phase of apparatus of Fig. 3, while liposome solution of L-α- dipalmitoyl phosphatidylcholine (DPPC) containing CoA was put in lower phase. Time course of the absorbance at 340 nm, which is assigned to the absorption of NADH, was measured. As shown in Fig. 4a, direct reaction of NADH and CoQ occurred after an increase in the concentration of NADH accompanying the permeation of NADH across the membrane. Then the repetition of increase and decrease of absorbance occurred, i.e., oscillation was observed. The decrease in pyruvate concentration led to the oscillation with short period and small amplitude after that of long period and large amplitude.
Secondly, we added cytochrome c and cytochrome c reductase to lower phase. The reaction in lower phase is thought to be equivalent to that of complex III in respiratory chain. In this reaction, the electron of NADH is transferred to cytochrome c via CoQ. As the wavelength of the absorption of NADH and of reduced type of cytochrome c is 340 nm and 550nm, respectively, the absorptions at 340nm and 550nm were measured at the same time. As shown in Fig. 4b, the oscillation of NADH appeared explicitly. Correspond to the oscillation of NADH, the oscillation of cytochrome c with same period was observed. Two oscillations were synchronized.
Furthermore, small quantity of cytochrome c oxidase was also added to lower phase. This reaction is thought of corresponding to that of complex IV in respiratory chain. As shown in Fig. 4c, the addition of cytochrome c oxidase led to little change of oscillation pattern at the same concentration as cytochrome c reductase. So, we reduced the concentration of cytochrome c reductase. As shown in Fig. 4d, when the concentration of cytochrome c oxidase equals to that of cytochrome c reductase, the oscillation with period of about 20 h occurred in both of NADH and cytochrome c. In greater quantity of cytochrome c oxidase than cytochrome c reductase, distinct oscillation with short period appeared. We found that the oscillatory reactions of NADH and CoQ induced continuous oscillatory reactions for the respiratory chain.
For a reaction using semipermeable membrane, we only obtained the oscillation with longer-period compared to the result with mitochondria. This may be owing to the shortage of supply of oxygen from the air required for the reaction of cytochrome c oxidase, because oxygen supply is hampered by the presence of upper phase sandwiched by dialysis membrane. Thus, in order to supply oxygen, to the system of Fig. 4c, 0.1 mL of 0.6% hydrogen peroxide was added to upper phase, while 0.1 mL of 0.1mg/mL catalase to lower phase. It has been found already that this condition leads no oscillation of oxygen 7. The result is shown in Fig. 4e. It was found that the short-period oscillation appeared by the supply of oxygen. Further increasing the concentration of cytochrome c reductase shortens the period of oscillation. Oxygen supply seems to facilitate the reaction of cytochrome c oxidase and to strengthen the oscillation.
The consecutive oscillations of intermediates in citric acid cycle caused by the oscillatory reaction of pyruvate dehydrogenase due to gradual entry of pyruvate
Pyruvate produced by glycolysis flows through mitochondrial membrane and is changed to acetyl-CoA by pyruvate dehydrogenase which catalyzes the reaction of pyruvate, NAD+ and CoA. When pyruvate passes across the mitochondrial inner membrane, mitochondrial pyruvate carrier (MPC), which is protein embedded within membrane, facilitates the pass of pyruvate across membrane 8–9. Since the flow of pyruvate is also considered to be caused by the difference of electrochemical potential between the inside and the outside of mitochondrial inner membrane, we might assume for simplicity that semipermeable membrane is able to be employed instead of mitochondrial membrane.
At first, in apparatus of Fig. 3, we put pyruvate solution in upper phase, while pyruvate dehydrogenase, CoA and NAD+ in lower phase, for measuring the formation of acetyl-CoA and NADH. Time course of the absorbance at 340nm due to the absorption of NADH was measured.
As shown in Fig. 5a, when the concentration of pyruvate was increased at the fixed concentrations of NAD+ and CoA, the period of NADH oscillation became short. On the other hand, decreasing the concentration of CoA at the fixed concentration of pyruvate and NAD+ led to a stepwise increase in absorbance (Fig. 5b). From these results, it was considered that the oscillation of acetyl-CoA, which was another product of this reaction, also happened.
As it was found that the formation of acetyl-CoA was oscillatory reaction in the mediation of semipermeable membrane, we conceived that subsequent reactions in citric acid cycle oscillate similarly. Therefore, oxaloacetic acid, citrate synthase and aconitase were also added to lower phase. The absorbance of at both 340nm and at 240nm, which were due to the absorptions of NADH and cis-aconitic acid, respectively, were measured at the same time.
As shown in Fig. 5c, not only the oscillation of NADH, but also that of cis-aconitic acid, the intermediate in the reaction of aconitase, was observed. When pyruvate concentration was small, the oscillations of NADH and cis-aconitic acid was restrained. However, as pyruvate concentration was increased, two oscillations were synchronized and the amplitude of cis-aconitic acid oscillation became large. Further increase of pyruvate led to short period and small amplitude oscillation of NADH. The oscillation of NADH is thought to induce the oscillatory reaction of aconitase via that of citrate synthase. The reactions of citric synthase and of aconitase are those in first stage of citric acid cycle. As cis-aconitic acid is the intermediate of citric acid cycle, this result suggests that the oscillatory reaction will be passed down to remained reactions in citric acid cycle.