Mechanism of action of human P-glycoprotein ATPase activity - Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites
Mechanism of action of human P-glycoprotein ATPase activity - Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites
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DOI：
10.1074/jbc.273.27.16631
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发表时间：
1998-07-03
影响因子：
4.8
通讯作者：
Gottesman, MM
中科院分区：
文献类型：
--
作者：
Hrycyna, CA;Ramachandra, M;Gottesman, MM
人P-糖蛋白（P-gp）是一种ATP依赖性的外排泵，负责人类癌症对多种亲脂性化合物的交叉抗性，由两个同源的半部分组成，每个半部分包含六个跨膜结构域和一个ATP结合/利用结构域。为了确定每个位点是否可以同时水解ATP，我们使用了原钒酸盐（Vi）诱导的ADP捕获技术（P-gp.MgADP.Vi）。与其他ATP酶类似，当分子与处于抑制催化过渡态的Vi（P-gp.MgADP.Vi）一起捕获并在紫外光存在下孵育时，在步行者A核苷酸结合结构域共有序列（GX（4）GK（T/S））内发生光化学肽键切割反应。重组成蛋白脂质体后，组氨酸标记的纯化的P-gp从杆状病毒感染的昆虫细胞具有药物刺激的ATP酶活性。将重构的P-gp与ATP或8-叠氮基-ATP在存在或不存在Vi的情况下在紫外线（365 nm）光下在冰上孵育60 min。将所得产物通过SDS-聚丙烯酰胺凝胶电泳分离，并用覆盖分子全长的7种不同的人P-gp特异性抗体进行免疫印迹。在不存在Vi的情况下，几乎没有观察到P-gp降解。在Vi的存在下，获得约28、47、94和110 kDa的产物，这与在任一ATP位点而不是两个位点处切割的预测分子量一致。在P-gp的接头区中的胰蛋白酶位点处或附近检测到另外的Vi依赖性切割位点。这些结果表明，氨基端和羧基端ATP位点都可以水解ATP。然而，没有证据表明ATE可以同时被两个位点水解。
Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX(4)GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites, An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino-and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATE can be hydrolyzed simultaneously by both sites.