| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
The maximum amount of acid-stable phosphoenzyme(E^<32>P)/mol of α-chain of pig gastric H/K-ATPase from [γ-^<32>P] ATP was found to be 0.5, which was half of that formed from ^<32>Pi. The maximum ^<32>P binding for the enzyme during turnover in the presence of [γ-^<32>P]ATP or [α-^<32>P]ATP was due to 0.5 mol of E^<32>P + 0.5 mol of an acid-labile enzyme-bound [γ-^<32>P]ATP (EATP) or 0.5 mol of an acid-labile enzyme-bound [γ-32P]ATP, respectively.
The turnover number of the enzyme (i.e.,the H^+-ATPase activity/(EP+EATP)) was very close to the apparent rate constants for EP breakdown and Pi liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of Pi liberated to that of EP that disappeared increased from 1 to 2 with increasing concentrations of ATP. This represents the first direct evidence, for the case of a P-type ATPase, in which 2 mol of Pi liberation occurs simultaneously from 1mol of EP for half of the enzyme molecules and 1 mol of EATP f
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or the other half during ATP hydrolysis.
Each catalytic α-chain is involved incross-talk, thus maintaining half-site phosphorylation and half-site ATP binding which are induced by high-and low-affinity ATP binding, respectively.
To characterize the oligomeric structure of H/K-ATPases, FITC-labeled H/K-ATPase molecules were observed by total internal reflection microscopy (TIRFM). Fluorescence images of a single fluorophore attached to protein, bound to a glass surface, were observed. The fluorescent image disappeared in a few seconds, indicating that photobleaching of the FITC occurs by laser irradiation. The photobleaching of FITC molecules appeared to be quantized. When the enzyme was solubilized by C12E8, protomeric and diprotomeric species were observed. On the other hand, solubilization was performed by n-octylglucoside, diprotomeric and tetraprotomeric species were mainly detected. Comparison of the enzymatic activity of solubilized materials, tetraprotomeric species are supposed to be a minimum essential unit of H/K-ATPase in the membrane. Less
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