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. 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-inactivation when temperature is scanned upward at 1 degree 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 degrees 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 degrees 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].(ABSTRACT TRUNCATED AT 250 WORDS)
Lepock
J R
JR
Guelph-Waterloo Program for Graduate Work in Physics, University of Waterloo, Ontario, Canada.
Rodahl
A M
AM
Zhang
C
C
Heynen
M L
ML
Waters
B
B
Cheng
K H
KH
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006358:Hot Temperature; D015394:Molecular Structure; D011489:Protein Denaturation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D013861:Thiocyanates; D014364:Tryptophan
D005452:Fluoresceins; D013861:Thiocyanates; D014364:Tryptophan; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium