[The role of histidine residues in conformational changes in the sarcoplasmic reticulum Ca-ATPase active site].
Conformational pH-induced changes of Mg-ATP binding site of the sarcoplasmic reticulum Ca-ATPase (SR-ATPase) were investigated by fluorescence energy transfer between covalently bound fluorescent label (fluorescein-5-isothiocyanate, FITC) and lanthanide ion (Nd3+). These changes were approximated by simple Henderson-Hasselbach equation with the apparent 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 to research the role of histidyl residues in this conformational transition. Diethyl pyrocarbonate was used to modify the histidyl residues of the SR-ATPase. The influence of histidyl modification on the functional parameters (the rates of ATP and p-nitrophenyl phosphate hydrolysis, the Ca transport and the level of Ca2+ accumulation) 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-dependent conformational changes in the active site of FITC-DEPC-SR-ATPase were studied by the method of the fluorescence energy transfer between FITC and Nd3+ in the region of pH 6-8. The histidyl modification of FITC-DEPC-SR-ATPase resulted in the significant shift of the curve of fluorescence energy transfer efficiency (the apparent pK > 7.5). These results suggest that the conformational transition in the active site of SR-ATPase was controlled by the histidyl residues.
Ivkova
M N
MN
Vinokurov
M G
MG
Pletnev
V V
VV
Pechatnikov
V A
VA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006639:Histidine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D004047:Diethyl Pyrocarbonate
Rol' gistidinovykh ostatkov v konformatsionnykh izmeneniiakh v aktivnom tsentre Ca-ATFazy sarkoplazmaticheskogo retikuluma.