4. A. V-type ATPases are capable of establishing a steep pH gradient across the

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4. A. V-type ATPases are capable of establishing a steep pH gradient across the membranes in which they are present. However, establishment of a steep pH gradient also produces a steep electrical gradient across that membrane as protons carry a positive charge. In biological situations, V-type ATPases are often found in the same membranes as ion channels that work to alleviate the electrical potential and thus maximize the pH gradient. You have a sample of a vacuole preparation from a plant source that is capable of establishing an intravacuolar (luminal) pH of 3.5 when incubated at 25°C in a pH 7.5 buffer system containing 3 mM MgATP and 50 mM KCl. However, when another sample of the same vacuole preparation is incubated in the same buffer system except that the KCl is replaced by 25 mM K2S04, the intravacuolar pH established is much higher and not much different from that of the pH 7.5 buffer system Provide an explanation for these results B. To your initial surprise, you discover that the proton motive force (pmf) across the vacuolar membrane established in the buffer system containing 25 mM K2SO4 is identical to the pmf established in the buffer system containing 50 mM KCI. How do you reconcile these findings? You should do calculations to make your point. C. Osteoclasts are specialized bone macrophages that resorb bone. In the figure below, an osteoclast is depicted. Bone maintenance requires osteoclasts to resorb certain regions of bone so that osteoblasts can re-form the mineralized matrix in that same region. Osteoclasts are multinucleated cells that bind to bone and form a resorption lacuna, an extracellular lysosome of sorts with specialized proteases and phosphatases that digest bone in an acidic compartment. The resorption lacuna is acidified by a V-type ATPase that moves protons across the osteoclast ruffled border membrane into the bone and allows for proteolytic digestion of bone. Note that an isozyme of carbonic anhydrase, carbonic anhydrase Il (CAIl), is expressed highly in osteoclasts. The ruffled border membrane contains a chloride channel (CICN7) and a V-type ATPase (TCIRG1) which is capable of pumping protons. The basolateral membrane contains a chloride-bicarbonate exchanger, similar to red blood cells. Explain how these enzymes and transporters work in concert to acidify the resorption lacuna. Explain also why loss of function mutations in TCIRG1, CICN7, or CAll all cause osteopetrosis, or 'stone bones'. Functional Secretory Domain CI Zone br, cr Bone Resorption Lacunae (pH-4.5 ' a.A Integrin ???, ici Exchanger Acidified VesiclesNucesPrytcEns

Explanation / Answer

Ans: A: To maintain the acidic ph 3.5 in the vacuole the ATPase has to pump in protons, in the presence of KCl normal activity is on hence ph is maintained. When KCl replaced with K2SO4, equilibrium is reached because ATPase has stopped working. In aqueous environment K2SO4 dissociates to 2K + SO4- as time passes, moles of SO4 increases and SO4 having the ability to bind to proteins and precipitate them, membrane proteins like ATPase cannot perform its normal function in the increasing concentration of SO4 ions hence equilibrium is reached (both vacuole and cytoplasm at same ph 7.5)

B. In terms of moles of K ions both 50mM KCl and 25mM K2SO4 having 50 mM of K (25XK2 = 50mM in K2SO4), hence initial pmf should be same, but in the case of K2SO4, SO4 ions arresting the ATPase and gradually pmf is affected.

C. To acidify resorption lacuna more protons should be pumped in to lacuna by ATPase this is coupled with Na and K pump. Increase in protons results in acidity and acidic proteases and phosphatases release carbonate and phosphate, increase in carbonate is coupled to chloride –bicarbonate exchanger (chloride in carbonate out)

Mutations in TCIRG1 results in no proton pumping in to lacuna, no increase in ph to operate acidic proteases and phosphatases.CLCN7 mutations can lead to no pumping in of chloride ions and bicarbonate pumping out is stopped, accumulation of bicarbonate leads to stone bones. Mutations in CAII fail to breakdown bicarbonate in to CO2 and H2O hence it accumulates.     

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