Functional interactions of catalytic site and transmembrane channel in the sarcoplasmic reticulum ATPase.
Vesicular fragments of longitudinal sarcoplasmic reticulum were loaded with calcium by active transport, sedimented by centrifugation, and resuspended in neutral buffer and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Under these conditions, calcium efflux from the loaded vesicles occurred at rates varying from 100 to 700 nmol/mg/min, depending on the calcium load. If either Ca2+ (microM), Mg2+ (mM), K+ or Na+ (greater than 10 mM) were added to the resuspension medium, the rate of efflux was reduced. In the presence of Mg2+ and EGTA, a large inhibition of calcium efflux was produced by formation of phosphoenzyme intermediate with Pi. In this case, addition of ADP again started calcium efflux, coupled with ATP synthesis. The rates of uncoupled or coupled efflux were approximately the same. The observed calcium fluxes are attributed to a slow channel formed by ATPase transmembrane helices (MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 686-700) and are capable of long range interaction with the catalytic site. Coupling of transport and catalytic activities is thereby produced by phosphorylation and ligand binding. The channel includes negatively charged residues that are likely to influence calcium fluxes through cation binding. It is proposed that this channel is the mechanistic device for active transport of calcium across the sarcoplasmic reticulum membrane, and for its reversal.
de Meis
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Departamento Bioquimica, Universidade Federal Rio de Janeiro, Brazil.
Suzano
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VA
Inesi
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology; D013463:Sulfuric Acid Esters / Q000494:pharmacology
D010710:Phosphates; D013463:Sulfuric Acid Esters; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D012964:Sodium; D000252:Calcium-Transporting ATPases; D008274:Magnesium; C007482:dimethyl sulfate; D011188:Potassium; D002118:Calcium