Abstract
The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.
Highlights
Ca2*uptakeinto vesicles reconstituted with enzyme alownaes stimulated2-2.5fold by the (Ca2+-Mg2+)-ATPase activator, added excompared properties of enzyme activity [13] with that of solubilized calcium transport component reconstituted into phospholipid vesicles [14]
The aim of the present study was to provide more definitive evidencethat thehigh affinity (Ca2’-Mg2’)-ATPase from liver plasma membranes is responsible for Ca2+transport.This was achieved by reconstitution experiments of the previously ogenously
On the basis of kinetic and biochemical studies, our group and several others [3,4,5,6,7,8,9,10,11] proposed thatthe high affinity (Ca2+-Mg2+)-ATPasien liver plasma membranes is responsible for Ca2+transport
Summary
In thispreparation the enzyme activity is purified about 20fold as compared to that in liver plasma membranes This the cell maintains the intracellularconcentration of free preparation is depleted in the activity of inhibitor protein, which we calcium (0.1 PM) 4 orders of magnitude lower than in the have previously purified and which confers enzyme sensitivity to extracellular medium (1-3 mM). This control is essential for inhibition by M$+ [16,17]. The activator proteinwas separated from normal cellular metabolism and for cellular responses to hormones or other external stimuli [1]
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