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
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.