Time-resolved charge translocation by sarcoplasmic reticulum Ca-ATPase measured on a solid supported membrane.
Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K(+) ions and at different pH values. Ca(2+) concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca(2+) concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca(2+) concentration, with a half-saturating value of approximately 1.5 microM. The pH dependence of the charge after Ca(2+) translocation demonstrates the occurrence of one H(+) per one Ca(2+) countertransport at pH 7 by direct charge-transfer measurements. The presence of K(+) decreases the magnitude of the current transients without altering their shape; this decrease is explained by K(+) binding to the cytoplasmic side of the pump in the E(1) conformation and being released to the same side during the E(1)-E(2) transition.
Tadini Buoninsegni
Francesco
F
Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italy.
Bartolommei
Gianluca
G
Moncelli
Maria Rosa
MR
Inesi
Giuseppe
G
Guidelli
Rolando
R
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000502:physiology*; D011188:Potassium / Q000502:physiology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D008051:Lipid Bilayers; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium