Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.
Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.
Yang
Su J
SJ
Department of Radiological Technology, Chungtai Institute of Health Sciences and Technology, Taichung 40605, Taiwan, ROC.
Jiang
Shih S
SS
Hsiao
Yi Y
YY
Van
Ru C
RC
Pan
Yih J
YJ
Pan
Rong L
RL
D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006358:Hot Temperature; D006868:Hydrolysis; D018546:Hypocotyl / Q000737:chemistry; D043564:Inorganic Pyrophosphatase / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification; D007700:Kinetics; D008962:Models, Theoretical; D008970:Molecular Weight; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011522:Protons; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D014617:Vacuoles / Q000201:enzymology*
D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011522:Protons; D043564:Inorganic Pyrophosphatase; D016650:Fluorescein-5-isothiocyanate