Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure.
Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.
Coelho-Sampaio
T
T
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
Ferreira
S T
ST
Benaim
G
G
Vieyra
A
A
D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D002147:Calmodulin / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006874:Hydrostatic Pressure; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D046911:Macromolecular Substances; D014638:Vanadates; D004492:Edetic Acid; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium