THE ROLE OF HISTIDYL RESIDUES IN CONFORMA TIONAL-CHANGES OF THE ACTIVE-SITE OF SARCOPLASMIC-RETICULUM CA-ATPASE

Citation
Mn. Ivkova et al., THE ROLE OF HISTIDYL RESIDUES IN CONFORMA TIONAL-CHANGES OF THE ACTIVE-SITE OF SARCOPLASMIC-RETICULUM CA-ATPASE, Biofizika, 41(1), 1996, pp. 86-94
Citations number
26
Categorie Soggetti
Biophysics
Journal title
ISSN journal
00063029
Volume
41
Issue
1
Year of publication
1996
Pages
86 - 94
Database
ISI
SICI code
0006-3029(1996)41:1<86:TROHRI>2.0.ZU;2-7
Abstract
Conformational pH-induced changes of Mg-ATP binding site of the sarcop lasmic reticulum Ca-ATPase (SR-ATPase) were investigated by fluorescen ce energy transfer between covalently bound fluorescent label (fluores cein-5-isothiocyanate, FITC) and lanthanide ion (Nd3+). These changes were approximated by simple Henderson-Hasselbach equation with the app arent pK 7,0+/-0,1 which is similar that of a histidyl residue [3]. In this work it was used the double chemical modification of SR-ATPase t o research the role of histidyl residues in this conformational transi tion. Diethyl pyrocarbonate was used to modify the histidyl residues o f the SR-ATPase. The influence of histidyl modification on the functio nal parameters (the rates of ATP and p-nitrophenyl phoshate hydrolysis , the Ca transport and the level of Ca2+ accumulaton) was monitored by the fluorescent probes (Quin-2, chlortetracycline) using fluorescent, spectrophotometric and pH-metric measurements. In the result of these experiments it was found the appropriate conditions to carry out the second modification. The DEPC-SR-ATPase was labeled by FITC. The pH-de pendent conformational changes in the active site of FITC-DEPC-SR-ATPa se were studied by the method of the fluorescence energy transfer betw een FITC and Nd3+ in the region of pH 6-8, The histidyl modification o f FITC-DEPC-SR-ATPase resulted in the significant shift of the curve o f fluorescence energy transfer efficiency (the apparent pK > 7,5). The se results suggest that the conformational transition in the active si te of SR-ATPase was controled by the histidyl residues.