Inhibition of ion pump ATPase activity by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe.
Inhibition of ion pump ATPase activity by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe.
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3-O-(4-苯甲酰基)苯甲酰基-ATP (BzATP) 对离子泵 ATP 酶活性的抑制：评估 BzATP 作为活性定点探针。
DOI：
10.1016/0005-2736(86)90492-x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Farley,RA
中科院分区：
文献类型：
--
作者：
Tran,CM;Farley,RA
本文研究了3‘-O-(4-苯甲酰基)苯甲酰三磷酸腺苷(BzATP)与RNAL(Na++K+)-ATPase、肌浆网钙转运ATPase和胃(H++K+)-ATPase的相互作用，以确定BzATP是否是从这些离子泵的ATP结合部位标记和鉴定多肽的合适探针。紫外光照射后，BzATP抑制了每个离子泵对ATP的酶解，并被共价掺入到每个蛋白质的100000道尔顿多肽中。反应溶液中过量的ATP既不能阻止ATPase活性的失活，也不能阻止BzATP对催化多肽的标记。然而，预先用荧光素-5‘-异硫氰酸酯(FITC)修饰ATPase，阻止了BzATP对100,000道尔顿多肽的大部分标记。BzATP竞争性地抑制了ATP与离子泵的高亲和力结合，但ATP不阻止酶对BzATP的高亲和力结合。BzATP与膜上的ATPase以高亲和力结合，其Kd值为0.8~1.2μM，aBmax为2~3nmoL/mg，也可与膜上至少一个低亲和力的高容量位点结合。从BzATP标记的(Na++K+)-ATPase的胰酶消化液中分离出的可溶性多肽表明，存在几个标记的多肽，其中没有一个是被ATP或FITC保护的。尽管BzATP可以从离子泵上的高亲和力结合部位取代ATP，因此，似乎酶活性的失活是BzATP与该部位以外的蛋白质反应的结果。因此，从这些实验中得出结论，BzATP不太可能是离子转运ATPase上的ATP结合位点的有用探针。
The interaction of 3′-O-(4-Benzoyl)benzoyl-ATP (BzATP) with the rnal (Na++ K+)-ATPase, the sarcoplasmic reticulum Ca-transport ATPase, and the gastric (H++ K+)-ATPase has been investigated in order to determine whether BzATP is a suitable probe for the labeling and identification of a peptide from the ATP binding sites of these ion pumps. After ultraviolet irradiation BzATP inhibited the enzymatic hydrolysis of ATP by each of the ion pumps, and also was covalently incorporated into the 100 000 dalton polypeptides of each protein. The presence of excess ATP in the reaction solution did not prevent either the inactivation of ATPase activity or the labeling of the catalytic polypeptides by BzATP. Prior modification of the ATPases with fluorescein-5′-isothiocyanate (FITC), however, prevented much of the labeling of the 100 000 dalton polypeptides by BzATP. BzATP competitively inhibited the high-affinity binding of ATP to the ion pumps, but ATP did not block the high-affinity binding of BzATP by the enzymes. BzATP binds to the membrane-bound ATPases at a high-affinity site with aKdof 0.8–1.2 μM and aBmaxof 2–3 nmol/mg, and also binds to at least one low-affinity, high-capacity site on the membranes. HPLC separation of the soluble peptides from a tryptic digest of BzATP-labeled (Na++ K+)-ATPase revealed the presence of several labeled peptides, none of which was protected by either ATP or FITC. Although BzATP can displace ATP from a high-affinity binding site on the ion pumps, it appears, therefore, that inactivation of enzymatic activity is the result of reactions between BzATP and the proteins at locations outside this site. Thus, it is concluded from these experiments that BzATP is not likely to be a useful probe for the ATP binding sites on the ion transport ATPases.