Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The site of interaction of dibucaine with the Ca(2+)-ATPase of rabbit sarcoplasmic reticulum, an ion-transporting membrane protein, was investigated by determining the effect of dibucaine on the denaturation of the transmembrane domain and the aqueous domain containing, respectively, the high-affinity Ca2+ binding sites and the site of ATP hydrolysis. In the absence of Ca2+, a single irreversible denaturation transition with Tm approximately equal to 49 degrees C is observed for the Ca(2+)-ATPase by differential scanning calorimetry (DSC). In the presence of Ca2+, but not Mg2+, Sr2+, or Ba2+, a new high-temperature transition is observed that has been shown to be due to stabilization of the transmembrane region [Lepock, J.

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate.

Thermal denaturation of the Ca2(+)-ATPase of sarcoplasmic reticulum reveals two 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.

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response.

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.