Investigation of reaction mechanism of gastric proton pump

胃质子泵反应机制研究

• 批准号: 13680703
• 负责人: KAYA Shunji
• 金额: $ 2.24万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680703/
• 关键词: Proton pump; subunit interaction; oligomeric structure; ナトリウムポンプ; 蛍光プローブ; 1分子観察; イオン輸送

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 … More 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

由[γ-^ P] ATP生成的酸稳定性磷酸酶（E^<32>P）/mol猪胃H/K-ATP酶α链<32>的最大量为0.5，是由^ Pi生成的最大量的一半<32>。在<32>[γ-^<32>P]ATP或[α-^<32>P]ATP存在下，在周转期间酶的最大^ P结合分别归因于0.5 mol E^<32>P + 0.5 mol酸不稳定酶结合的[γ-^<32>P]ATP（EATP）或0.5 mol酸不稳定酶结合的[γ-32 P]ATP。H^+-ATP酶活性/（EP+EATP））与EP分解和Pi释放的表观速率常数非常接近，二者均随ATP浓度的增加而降低。随着ATP浓度的增加，释放的Pi量与消失的EP量之比从1增加到2。这代表了P型ATP酶的第一个直接证据，在这种情况下，对于一半的酶分子，2摩尔的Pi同时从1摩尔的EP和1摩尔的EATP中释放出来。 ...更多信息 为了表征H/K-ATP酶的寡聚体结构，用全内反射显微镜（TIRFM）观察了H/K-ATP酶的荧光标记，并对H/K-ATP酶的荧光结构进行了分析。观察到与玻璃表面结合的蛋白质连接的单个荧光团的荧光图像。荧光图像在几秒钟内消失，表明激光照射发生了FITC的光漂白。FITC分子的光漂白似乎是量子化的。当酶被C12 E8增溶时，观察到原聚体和双原聚体物种。另一方面，正辛基葡萄糖苷进行增溶，主要检测到二元体和四元体物种。通过比较溶解物质的酶活性，四元体被认为是膜中H/K-ATP酶的最小必需单位。少

项目成果

K. Taniguchi: "The Oligomeric Nature of Na/K-Transport ATPase"Journal of Biochemstry. 129. 335-342 (2001)

K. Taniguchi：“Na/K-转运 ATP 酶的寡聚性质”生物化学杂志。

N. Fujitani et al.: "Structure determination and Conformational change induced by tyrosine phosphorylation of the N-termimal domain of the α-chain of pig gastric H^+/K^+-ATPase"Biochem, Biopys. Res. Commun.. 3, 300(1). 23-229 (2003)

N. Fujitani 等人：“猪胃 H^+/K^+-ATP 酶 α 链的酪氨酸磷酸化诱导的结构测定和构象变化”Biochem，Biopys Res。 3、300(1)。23-229(2003)。

K.Abe: "Gastric H/K-ATPase liberates two moles of Pi from one mole of phosphoenzyme fromed from a high-affinity ATP binding site and one mote of enzyme-bound ATP at the low affinity site during cross-talk between catalytic subunits"Biochemstry. 192・41(2).

K.Abe：“在催化亚基之间的串扰过程中，胃 H/K-ATP 酶从高亲和力 ATP 结合位点的 1 摩尔磷酸酶和低亲和力位点的酶结合 ATP 中释放出 2 摩尔 Pi”生物化学。192・41(2)。

S.Kaya: "Oligomeric structure of P-type ATPases observed by single molecule detection technique"Ann. N. Y. Acad. Sci.. 986(印刷中). (2003)

S.Kaya：“通过单分子检测技术观察到的 P 型 ATP 酶的寡聚结构”Ann. N. Y. Sci.. 986（出版中）。

N.Fujitani: "Structure determination and Conformational change induced by tyrosine phosphorylation of the N-terminai domain of the α-chain of pig gastric H^+/K^+-ATPase"Biochem. Biopys. Res. Commun.. 3・300(1). 223-229 (2003)

N.Fujitani：“猪胃H^+/K^+-ATP酶的N-末端结构域的酪氨酸磷酸化诱导的结构测定和构象变化”Biochem.3・300。 (1)。223-229(2003)。