1A. V-ATPases pump protons inside vacuoles. If we imagine this transport was isolated, it would result in a pH gradient and significant charge difference across the vacuolar membrane. As this transport takes place in the context of living organelles, ion channels facilitate the transport of ions across the same membrane down the electrical gradient that arises as a result of V-ATPase mediated active transport of protons. Thus, movement of ions enables pH gradient and maintaining of low-pH conditions inside vacuole.
In the experiment presented here, the change of anion present in the solution surrounding vacuole preparation retards the proton pumping inside the vacuole. This suggests that the main force maintaining membrane potential is not potassium transport outside the vacuole (in the opposite direction of proton movement), but the influx of chloride ions through chloride ion channels. Movement of an equimolar amount of protons inside vacuole, and chloride iones outside vacuole would result in maintaining both, membrane potential, and the pH gradient established by V-ATPase. On the other hand, the membrane is likely to lack potassium and sulfide ion channels that could take the role of chloride in the case of K2SO4 presence. Or they are present but not in the same amount and with the same role of maintaining low pH inside vacuole.
1B. Proton motive force as the energy of the proton concentration gradient is affected by chemical (pH difference, ΔpH) and electric potential (charge difference, Δψ). Mathematically expressed, it is:
Δp= Δψ-0.059 x ΔpH
In case 1, when vacuoles are in the buffer containing 50 mM KCl, and in case 2, when vacuoles are in the buffer contacting 25 mM K2SO4, proton motive forces are equal:
Δp1=Δp2
Δψ1-0.059 V x ΔpH1=Δψ2-0.059 V x ΔpH2
As pH difference in the case 1 is known to be 4 and pH difference in case 2 is known to be about 0, so the expression becomes:
Δψ1-0.059 V x 4=Δψ2-0.059 V x 0
Δψ1-0.059 V x 4=Δψ2-0.059 V x 0
Δψ1-0.236 V=Δψ2
This means that in the second case, where K2SO4 is present around vacuoles, the absence of chloride ions stops proton movement through V-type ATPase. Anyway, proton motive force is still the same suggesting (as seen from the calculation) that the electrical potential is very high and protons are highly likely to leave the vacuole. As pH is the same as in the surrounding solution, there must be another ion movement resulting in charge difference. The only possible movement in these controlled conditions that can contribute to the described charge separation is potassium. These data then suggest that, in the presence of high chloride concentration (higher than normal in intracellular space where vacuoles are located), V-type ATPase pumps even more protons to the inside of vacuole resulting in high pH difference, higher than common for physiological conditions (where vacuolar pH is close to 5). This means that in any case – in the presence of KCl, and in the presence of K2SO4 (complete absence of chloride) transport of ions in and out vacuoles is disrupted. In the case chloride is absent V-ATPase potassium enters the cell to a high level. Chloride and proton movements are highly intertwined and control potassium transport. In the total absence of chloride, protons are likely (according to PMF) to leave the vacuole even though the pH is neutral to alkaline, which is not a physiological state for a vacuole either.

1C. As resorption lacuna requires acidic pH for enzymatic bone resorption, V-type ATPase (TCIRG1) is highly expressed on the ruffled border side of an osteoclast. TCIRG1 pumps protons inside this extracellular lacuna. To maintain the membrane potential of osteoclast, chloride is transported through chloride channels (ClCN7) in

2. An enzyme with a Kn of 2.4x10&quot;4 M was assayed at the follo...

2. An enzyme with a Kn of 2.4x10"4 M was assayed at the follo...