title: Conformational differences between the E1 and E2 states of the calcium adenosinetriphosphatase of the erythrocyte plasma membrane as revealed by circular dichroism and fluorescence spectroscopy.
abstract: Different conformational states of the purified plasma membrane Ca2+-ATPase from pig erythrocytes have been detected by circular dichroism (CD) and fluorescence spectroscopy. The helical content of the enzyme decreased by about 10% in the transition from the Ca2+ high-affinity form (10 microM free Ca2+ = E1 state) to the VO4(3-)-inhibited state (20 microM VO4(3-) = E2 state). The changes in the CD spectra did not show full reversibility upon reversing the E1-E2 transition, whereas those in the fluorescence spectra did. A temperature-dependent loss of alpha-helical content in the presence of Ca2+ was also observed. Intrinsic fluorescence measurements revealed an increase in fluorescence intensity upon addition of Ca2+. The change was fully reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The increase in fluorescence intensity was partly reversed by adding ATP, an effect which is suggested to correspond to the "Ca2+-occluded" form of the ATPase. The steady-state level of the fluorescence intensity was stable for several minutes in the presence of 100 microM ATP. By contrast, the decrease of fluorescence intensity induced by limiting concentrations of ATP (= 1 microM) was only transient, indicating the decomposition of the phosphorylated intermediate of the ATPase and the reestablishment of the Ca2+ high-affinity form of the enzyme.
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013057:Spectrum Analysis; D013552:Swine / Q000097:blood; D013696:Temperature; D014638:Vanadates / Q000494:pharmacology
chemical_list: D014638:Vanadates; D000252:Calcium-Transporting ATPases