ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.
ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.
复制标题
大脑 p97/VCP（含缬氨肽蛋白）中的 ATP 结合位点，一种多功能 AAA ATP 酶。
作者：
R. Zalk;V. Shoshan
VCP（valosin-containing protein）或p97是AAA家族（与多种细胞活性相关的ATP酶家族）的成员，AAA家族是一组不同的蛋白质，其共享包含重复的推定ATP结合位点的关键保守AAA模块。虽然AAA家族的功能与其假定的ATP结合位点有关，但ATP与这些位点的结合尚未得到证实。在本研究中，使用光反应性ATP类似物BzATP [3 '-O -（4-苯甲酰基苯甲酰基）ATP]表征脑VCP的ATP结合位点。Bz-[alpha-（32）P]ATP的光活化导致其与97 kDa纯化的可溶性或膜相关蛋白（通过氨基酸测序鉴定为VCP）共价结合。Bz-[α-（32）P]ATP以表观高亲和力（74-111 nM）共价结合至纯化的同源六聚体VCP。使用不同的放射性标记分析和蛋白质测定方法，测定每个同源六聚体VCP（n =6）结合的2.23+/-0.14 BzATP的摩尔化学计量。核苷酸抑制Bz-[α-（32）P]ATP与VCP结合的效率为：BzATP>ATP>ADP>>腺苷5 '-[β，γ-亚氨基]三磷酸>或=腺苷5'-[β，γ-亚甲基]三磷酸，而AMP、GTP和CTP则无效。观察到VCP具有非常低的ATP酶活性，其核苷酸特异性与BzATP结合的核苷酸特异性相似。由ATP结合的交替位点机制诱导的构象变化被认为是将ATP结合与AAA家族的不同活性偶联的分子机制。
VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family.
登录
查看更多内容
影响因子：
5.6
作者：
Putnam, CD;Clancy, SB;Tainer, JA
通讯作者：
Tainer, JA
影响因子：
3.1
作者：
Hongtao Zhang;Qiang Wang;K. Kajino;Mark I. Greene
通讯作者：
Hongtao Zhang;Qiang Wang;K. Kajino;Mark I. Greene
26 S 蛋白酶的亚基 4 是新型真核 ATP 酶家族的成员。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Dubiel,W;Ferrell,K;Pratt,G;Rechsteiner,M
通讯作者：
Rechsteiner,M
影响因子：
16
作者：
Leonhard, K;Guiard, B;Langer, T
通讯作者：
Langer, T