Isolation and Preliminary Characterization of ATPase from Olive Calli Grown at Different Auxin/Cytokinin Ratio

ATPase activity was studied in calli from olive (Olea europaea L.) petioles cultured in media modified in their auxin/cytokinin ratio in order to induce different morphogenetic responses. Addition of 0.54 μM α-naphthaleneacetic acid (NAA) or 14 μM zeatin (ZEA) did not induce any morphogenesis in calli and proton pump activity in vivo was very low, while calli produced roots at 27 or 11 μM NAA + 0.28 μM ZEA and possessed clearly detectable proton pump activity. ATPase activity associated with microsomes isolated by differential centrifugation from different callus cultures had the same pH optimum and similar sensitivity toward nitrate and azide. However, microsomes isolated from non-morphogenetic calli had higher specific ATPase activity which was very poorly (6 %) inhibited by vanadate. Also, the fractionation of these microsomes on a continuous sucrose gradient showed two peaks of ATPase activity, the more pronounced one co-purifying with the Golg i marker enzyme, Triton-stimulated UDPase activity, suggesting thus the presence of very high ATPase activity in Golgi secretory vesicles. On the contrary, ATPase activity of microsomes from calli producing roots was more sensitive to vanadate (30 - 40 % inhibition). Furthermore, the component of ATPase activity attributable to Golgi secretory vesicles was less abundant. This revised version was published online in July 2006 with corrections to the Cover Date.