title: Two-step internalization of Ca2+ from a single E approximately P.Ca2 species by the Ca2+-ATPase.
abstract: Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP.Ca2 obtained by rapid mixing of E.Ca2 with [gamma-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to approximately 34 s-1, followed by EP* decay with a rate constant of approximately 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP.Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approximately P.Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 microL/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].
authors.affiliation: Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
authors.forename: D
authors.initials: D
authors.lastname: Khananshvili
authors.forename: W P
authors.initials: WP
authors.lastname: Jencks
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D000252:Calcium-Transporting ATPases; D002118:Calcium