THERMAL-DENATURATION OF THE CA-2+-ATPASE OF SARCOPLASMIC-RETICULUM REVEALS 2 THERMODYNAMICALLY INDEPENDENT DOMAINS

Inactivation of Ca2+ uptake and ATPase activity of the Ca2+-ATPase of rabbit sarcoplasmic reticulum was measured and compared to the thermal denaturation of the enzyme as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. Two fluorophores were monitored: intrinsic tryptophan (localized in the transmembrane region) and fluorescein isothiocyanate (FITC)-labeled Lys-515 (located in the nucleotide binding domain). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Activation energies (EA) and frequency factors (A) for inactivation were obtained for the enzyme in 1 mM EGTA and 1 mM Ca2+. These were transformed to a transition temperature for inactivation, Tm (defined as the temperature of half-in-activation when temperature is scanned upward at 1 °C/min). All denaturation profiles were fit with an irreversible model to obtain EA and Tm for each transition, and the values of these parameters for denaturation were compared to the values for inactivation. In EGTA, denaturation obeys a single-step model (Tm = 49 °C), but a two-step model is required to fit the DSC provile of the enzyme in 1 mM Ca2+. The specific locations of tryptophan and the fluorescein label were used to demonstrate that denaturation in Ca2+ occurs through two distinct thermodynamic domains. Domain I (Tm = 50 °C) consists of the nucleotide binding region and most likely the phosphorylation and transduction regions [MacLennan, D. H., Brandl, C. J., Korczak, B., & Green, N. M. (1985) Nature 316, 696–700]. Domain II (Tm = 57–59 °C) consists of the transmembrane region and probably the stalk region. Inactivation of ATPase activity and Ca2+ uptake when heated in 1 mM Ca2+ is due to denaturation of domain I. Inactivation of ATPase activity (Tm = 49 °C) when heated in 1 mM EGTA is also due to denaturation of domain I, but inactivation of Ca2+ uptake (Tm = 37 °C), yielding uncoupling of Ca2+ uptake from hydrolysis of ATP, does not correlate with any conformational change in the Ca2+-ATPase detectable by these methods. From these results, it can be inferred that the conformational change occuring during the E2 → E1 transition occurs primarily in domain II (ΔTm = 8−10 °C), although there is a small stabilization of domain I (ΔTm = 1 °C), demonstrating interaction and communication between these domains which is necessary for Ca2+-dependent ATPase activity. © 1990, American Chemical Society. All rights reserved.