Abstract
The vacuole is a fundamental, domainant and primitive organelle and occupies a large part of the total cell volume in most mature plant cells. Higher plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on its membrane. V-ATPase is widely distributed in all eukaryotic cells, V-PPase has been found only in plants and a photosyntheic bacterium. It is extensively known that these two enzymes generate roughly equal proton gradients cross the vacuolar membrane (tonoplast). Mean while, both pumps respond to stress in order to maintain the critical functions of the vacuole. In this work, a vacuolelemma-enriched fraction from etiolated mung bean seedlings (Vignaitalic radiata L.) were used to investigate the function of these two enzymes upon shape change of vacuolar membrane. We utilized sucrose and sorbitol (upto 1 M) to induce swell and shrink of vacuole, respectively. Under high sucrose or sorbitol, proton translocation and specific activity of these two enzymes were inhibited to different extent. Changes of Vmax and Km of vacuolar H+-ATPase under sucrose implied an increase in substrate-binding affinity, but a decrease in the substrate hydrolysis activity, respectively. And the vacuolar H+-PPase under sorbitol or sucrose treatment showed slightly the same. However, sorbitol treatment decreased Vmax of the vacuolar H+-ATPase without changed its affinity to substrate. We suggested that the membrane environment may play an important role in proton pumping systems.It is possible that, following membrane swelling, structure of the proton pores of these two proton enzymes was changed. And following membrane shrinkage, the catalytic site of enzymes indicates a possible conformational change. Therefore, working models are proposed to interpret the membrane effects of vacuolar H+-PPase and H+-ATPase.Abbreviations ---------------------------------------------------------- 1Abstract ----------------------------------------------------------------- 3Introduction ------------------------------------------------------------ 7Materials and Methods ----------------------------------------------- 18Results ------------------------------------------------------------------ 29Discussion -------------------------------------------------------------- 36References -------------------------------------------------------------- 42Figures & Tables ------------------------------------------------------- 53