title: Susceptibility test of two Ca(2+)-ATPase conformers to denaturants and polyols to outline their structural difference.
abstract: 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 Ca(2+)-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.
authors.affiliation: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, 6-3 Aza Aoba, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kotake
authors.forename: Genichi
authors.initials: G
authors.lastname: Tajima
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Maruyama
authors.forename: Jun
authors.initials: J
authors.lastname: Nakamura
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D000818:Animals; D019791:Guanidine / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D011108:Polymers / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
chemical_list: D011108:Polymers; C024617:polyol; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019791:Guanidine