Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Javier Navarro, A. Essig

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate CA2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to AtP hydrolysis of }1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca 1/2 ± transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations ≤2 μM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradient and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.

Original languageEnglish (US)
Pages (from-to)709-717
Number of pages9
JournalBiophysical Journal
Volume46
Issue number6
StatePublished - 1984
Externally publishedYes

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Calcium-Transporting ATPases
Hydrolysis
Adenosine Triphosphate
Valinomycin
Density Gradient Centrifugation
Ionophores
Quercetin
Calcimycin
Octoxynol
Sarcoplasmic Reticulum
Detergents
Membrane Potentials
Sucrose
Phospholipids
Potassium
Coloring Agents
Cholesterol
Lipids
Light
Membranes

ASJC Scopus subject areas

  • Biophysics

Cite this

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles. / Navarro, Javier; Essig, A.

In: Biophysical Journal, Vol. 46, No. 6, 1984, p. 709-717.

Research output: Contribution to journalArticle

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