Binding of calcium to the calcium adenosine triphosphatase of sarcoplasmic reticulum

Abstract

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.

A Remarkably Stable Phosphorylated Form of Ca2+-ATPase Prepared from Ca2+-loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles

“…We found that the rate constant of the fluorescence rise was about 10 s Ϫ1 at 30 M free Ca 2ϩ (pCa 4.5), and this was almost the maximal value (the rates measured at 300 M and 2 mM free Ca 2ϩ were 13 and 15 s Ϫ1 , respectively). This rate was of the same order of magnitude as that for Ca 2ϩ binding to dephosphorylated native ATPase in the absence of nucleotides under similar conditions (19,20). The Ca 2ϩ dependence of the stability of the low fluorescence species was qualitatively similar after collapsing the calcium gradient by either ionophore or detergent (data not shown).…”

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

“…Investigation ofthe partial reactions of the pump cycle has shown that translocation of Ca2" takes place in the first part of the cycle and can be divided into three main steps, involving binding on the cis side of the membrane, occlusion within the protein, and release to the trans side or vesicle lumen (1)(2)(3)(4)(5)(6)(7). These events at the transport site are coupled to chemical changes at the active site such as the change in the reactivity of an aspartic residue to ATP, phosphorylation of the residue to an ADPsensitive form, and then a change to an ADP-insensitive phospho form (8)(9)(10).…”

“…These intermediates are those with the Ca21 sites oriented toward the vesicle lumen (13). In the forward direction of catalysis, the block forces the enzyme to follow the uncoupled pathway, involving direct hydrolysis of the ADP-sensitive E1-P(2Ca) intermediate (step 7).…”

Ca2+ Release to Lumen from ADP-sensitive Phosphoenzyme E1PCa2 without Bound K+ of Sarcoplasmic Reticulum Ca2+-ATPase

“…In E1Ca 2 , Ca 2ϩ bound at site II is rapidly exchanged with the cytoplasmic Ca 2ϩ , and the Ca 2ϩ bound at the deeper site I can be released to the cytoplasm only when site II is vacant (15,(41)(42)(43)(44). Therefore, we first labeled site I with 45 Ca 2ϩ by exchanging the site II-bound 45 Ca 2ϩ with nonradioactive Ca 2ϩ (supplemental Fig.…”

“…due to Ca 2ϩ exchange with site II (see supplemental Fig. S1) (42)(43)(44). Then, 45 Ca 2ϩ uptake assay in a single turnover was performed as in Fig.…”