H Weissbach, B Redfield, N Qiu, G Chen, A Carlino, V Vidal, O Tsolas, N Brot
{"title":"Interaction of BiP with substance P and nucleotides.","authors":"H Weissbach, B Redfield, N Qiu, G Chen, A Carlino, V Vidal, O Tsolas, N Brot","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>A rapid and simple spin column assay has been used to study interactions of BiP with substance P (SP) and ATP. At 4 degrees C, the binding of SP to BiP requires ATP and a stable SP-BiP.ATP complex is formed. Nonhydrolyzable ATP analogues or ADP cannot replace ATP. Although ATP converts BiP dimers to monomers, the requirement for ATP for SP binding is not solely due to BiP dissociation, because purified BiP monomers also require ATP for peptide binding. At 37 degrees C, there is rapid binding of SP to BiP even in the absence of ATP and, in fact, ATP at concentrations above 5 microM causes release of SP from BiP. At this higher temperature, there is also rapid hydrolysis of ATP bound to BiP. These results extend our previous results (Brot et al., 1994) that indicated the formation, at low ATP concentrations, of a labile SP.BiP.ATP complex that, after ATP hydrolysis, resulted in a stable SP.BiP.ADP complex.</p>","PeriodicalId":72545,"journal":{"name":"Cellular & molecular biology research","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cellular & molecular biology research","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
A rapid and simple spin column assay has been used to study interactions of BiP with substance P (SP) and ATP. At 4 degrees C, the binding of SP to BiP requires ATP and a stable SP-BiP.ATP complex is formed. Nonhydrolyzable ATP analogues or ADP cannot replace ATP. Although ATP converts BiP dimers to monomers, the requirement for ATP for SP binding is not solely due to BiP dissociation, because purified BiP monomers also require ATP for peptide binding. At 37 degrees C, there is rapid binding of SP to BiP even in the absence of ATP and, in fact, ATP at concentrations above 5 microM causes release of SP from BiP. At this higher temperature, there is also rapid hydrolysis of ATP bound to BiP. These results extend our previous results (Brot et al., 1994) that indicated the formation, at low ATP concentrations, of a labile SP.BiP.ATP complex that, after ATP hydrolysis, resulted in a stable SP.BiP.ADP complex.
采用快速简便的自旋柱法研究了BiP与P物质(SP)和ATP的相互作用。在4℃时,SP与BiP的结合需要ATP和稳定的SP-BiP。形成ATP复合物。不可水解的ATP类似物或ADP不能取代ATP。虽然ATP将BiP二聚体转化为单体,但SP结合对ATP的需求并不仅仅是由于BiP解离,因为纯化的BiP单体也需要ATP来结合肽。在37℃时,即使没有ATP, SP也能迅速与BiP结合,事实上,浓度高于5微米的ATP会导致SP从BiP中释放出来。在这个更高的温度下,与BiP结合的ATP也会快速水解。这些结果扩展了我们之前的结果(Brot et al., 1994),表明在低ATP浓度下,形成不稳定的SP.BiP.ATP复合物,在ATP水解后,形成稳定的SP.BiP.ADP复合物。