| The unicellular green alga Dunaliella salina lives in the Dead Sea and grows optimally at high concentrations of salt. However, the cytosolic concentration of Na+ is comparable to that in other organisms, and purified proteins from Dunaliella are not active under high-salt conditions. Therefore there must be a mechanism to remove Na+ from its intracellular space. Although the Na +/H+ antiporter carries out this task in many organisms, it is not involved in Dunaliella because I found that its inhibitor did not affect several vital reactions, including swimming, respiration, and photosynthesis. Moreover, based on a thermodynamic analysis, the antiporter, with known coupling ratios, is not able to extrude Na+ externally. As a possible explanation, I postulated the presence of a Na+-ATPase at the plasma membrane.; In order to explore the presence and role of a plasma membrane Na +-ATPase in Dunaliella, I initially set out to purify the plasma membrane. However, probably due to the unique environment in which this alga lives, conventional methods such as aqueous polymer two-phase partitioning or density gradient centrifugation did not work. Two findings changed my focus to start purifying the ATPase from the microsomal fraction: first, I detected a Na+-stimulated vanadate-sensitive ATPase activity from flagellar membrane; second, I learned that membrane cycling at the plasma membrane is very rapid and frequent in Dunaliella, hence blurring the distinction between the plasma membrane and endomembranes. Therefore, I decided to purify the ATPase from the microsomal fraction in order to obtain a quantity sufficient to characterize it biochemically. Because the combination of ion exchange and gel filtration chromatography alone reported by many to purify plant plasma membrane ATPases gave poor results, I applied a variety of column chromatographic techniques to develop a reliable and efficient purification procedure. In addition to ion exchange and gel filtration chromatography, I tested many extraction and fractionation methods as well as hydrophobic interaction chromatography, affinity chromatography, and chromatofocusing under numerous conditions. Using both a ConA column and a ceramic hydroxyapatite column, I was able to partially purify an ATPase. The specific activity was increased by 2000 fold compared to the microsomal fraction and the ATPase was sensitive to vanadate and 100 kD in size. |
