To determine the effect of denaturants [guanidine hydrochloride (GdnHCl) and urea] and polyols [with various molecular masses (62.1–600)] on calcium binding at the two hypothesized conformers (A and B forms) of the chemically equivalent sarcoplasmic reticulum Ca2+-ATPase, which bind two calcium ions in different manners, we examined the effect of these reagents on the calcium dependence of ATP-supported phosphorylation of the ATPase molecules and of their calcium-activated, acetyl phosphatate hydrolytic activity. (1) GdnHCl (~0.05 M) and urea (~0.5 M) increased the apparent calcium affinity (K 0.5) of 2–6 μM of noncooperative binding [Hill coefficient (n H) ~ 1] of the A form to 10–40 μM. (2) The employed polyols transformed the binding of the A form into cooperative binding (n H ~ 2), accompanying the approach of its K 0.5 value to that (K 0.5 = 0.04–0.2 μM) of the cooperative binding (n H ~ 2) of the B form; the transition concentration (0.025–2 M) of the polyols, above which such transformation occurs, was in inverse relation to their molecular mass. (3) The binding of the B form was resistant to these denaturants and polyols. Based on these data, a structural model of the two forms, calcium-binding domains of which are loosely and compactly folded, is presented.
The Journal of Membrane Biology – Springer Journals
Published: Nov 3, 2012
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