Abstract
Cys674 of the sarcoplasmic reticulum Ca2+-ATPase was selectively labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and the steady-state fluorescence anisotropy of this label and its total fluorescence intensity were followed throughout the catalytic cycle. At 25 degrees C, the anisotropy and the total fluorescence intensity increased by 2.1 and 9.4%, respectively, upon Ca2+ binding to the high affinity sites. Upon subsequent ATP binding to the catalytic site, the anisotropy and the total fluorescence intensity decreased by 6.8 and 23.9%, respectively. These drops likely occurred in the enzyme.ATP complex. The extents of changes upon additions of Ca2+ and ATP in the anisotropy, but not in the total fluorescence intensity, were greatly reduced by lowering the temperature. Slight drops in the anisotropy and the total fluorescence intensity occurred upon conversion of phosphoenzyme (EP) from the ADP-sensitive form to the ADP-insensitive form. The anisotropy and the total fluorescence intensity returned to the initial level when EP was hydrolyzed. Mg2+-dependent Pi-induced drops in the anisotropy and the total fluorescence intensity occurred coincidently with EP formation from Pi. These demonstrate that the ATP-induced drops in the anisotropy and the total fluorescence intensity are predominant throughout the catalytic cycle. Most probably, the changes in the anisotropy are due to changes in the rotational diffusion of the label. These findings indicate that ATP binding to the catalytic site induces a relaxed conformation in the microenvironment of the label bound to Cys674.
Highlights
CYS“~of the sarcoplasmic reticulum Ca2+-ATPase sensitive EP,’ which can react with added ADP to form ATP
At 25 “C, revealed, andthe secondary structureand the functional the anisotropy and the totafluorescence intensity in- domains were deduced on the basis of this primary structure 2btceion3rnientg.zaad9ylts%iomen,fdgtelhru-teebAoosyrptTe2hhe.sPiec1cgtcechiaanvontcmeaadelflypfy9il.iten.ni4xicTt%t.eyhn,Tessrsshieiiettetsyeedpesd,ee.xrcctotUhrtepieepvnasaoetssnnlelyiidbko,ssofyueultbcyprsohoepanoqnycugcCea6eunn.asr8td2ruA+etphdaTtonehbPndieinnd-tstdd(hhi5yogeeF)nmn.raesiIaofmunniimbznciytmacshtmnhieobntielesleahtowcthaastuseeavsplsmiacoeorsafcpreltttteal,ohevltfiyecneottltici,heocvailueeuttpralcirwylsrioyedelcntaclaielobectswenedsalte.tecihfemdtieixnvwppeitoetdriytrarhi,tsnmaiasInt-neeptEndotitDrnosttAhi(mtn6ehNve,efS7celuhsf)wotua,irnngietciahssttcomieoeutnntohoacnaefle additions of Ca2+and ATP in the anisotropy, nboutt in intensity of the bound EDANS was followed throughout the the total fluorescence intensity, were greatly reduced catalytic cycle
Slight drops in the ani-Ca2+binding to the high affinity sites and greatly decreased sotropy and the total fluorescence intensity occurred upon ATP binding to the catalytic site
Summary
EDANS-The emission spectrum of the total fluorescence intensity of the bound EDANS was determined at 25 "C Saturating concentration of Ca2+caused an increase in the fluorescence intensity by 9.4% and anincrease in the anisotropy by 2.1%.When ATP was added to give 10 PM after the addition ofCaC12, the fluorescence intensity and the anisotropy decreased by 23.9 and 6.8%,respectively.They returned spontaneously to theinitial levels, but therestoration of the fluorescence intensity was incomplete. This incomplete restoration is presumably due to an effect of ADP which was formed from ATP. The totalfluorescenceintensity and Changes in the Total Fluorescence Intensity and Fluores- the anisotropy were calculated using the observed G factor of cence Anisotropy ofthe Bound EDANS following Additions of 0.571.
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