| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 91 === ABSTRACT
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role to maintain the acidity of cytoplasm via the proton translocation from cytosol to vacuolar lumen at the expense of PPi hydrolysis. A line of evidence indicates that the carboxyl terminus of V-PPase is close to the catalytic site. Investigation in our laboratory disclosed that the removal of C-terminus in V-PPase by carboxylpeptidase A released the stimulation of K+. In this study, we attempted to identify the roles of the C-terminus of V-PPase by C-terminus deletion. A serious of C-terminus deleted mutants were constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Our results indicated that C-terminus deleted 5 amino-acid mutant (△C5) induced a decline in enzymatic activity, proton translocation, and coupling ratio of V-PPase, but C-terminus deleted 10 amino-acid mutant (△C10) had enzymatic activity about 60-70% of control. The mutant △C10 shifted T1/2 (pretreatment temperature at which half enzymatic activity is observed) but not the optimum pH for PPi hydrolytic activity. The deletion of C-terminus relieved the stimulation of K+, implying that the C-terminus is most likely the candidate for the region involved in the K+-binding and/or probably the K+-regulation of the enzyme.
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