Biophysical Journal 72: 2503-2514 (1997)
© 1997 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hartung, K Articles by Fendler, K Search for Related Content PubMed PubMed Citation Articles by Hartung, K Articles by Fendler, K

Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump.

Max-Planck-Institut für Biophysik, Kennedyallee, Frankfurt/Main Germany.

ABSTRACT

Time-resolved measurements of currents generated by Ca-ATPase from fragmented sarcoplasmic reticulum (SR) are described. SR vesicles spontaneously adsorb to a black lipid membrane acting as a capacitive electrode. Charge translocation by the enzyme is initiated by an ATP concentration jump performed by the light-induced conversion of an inactive precursor (caged ATP) to ATP with a time constant of 2.0 ms at pH 6.2 and 24 degrees C. The shape of the current signal is triphasic, an initial current flow into the vesicle lumen is followed by an outward current and a second slow inward current. The time course of the current signal can be described by five relaxation rate constants, lambda1 to lambda5 plus a fixed delay D approximately 1-3 ms. The electrical signal shows that 1) the reaction cycle of the Ca-ATPase contains two electrogenic steps; 2) positive charge is moved toward the luminal side in the first rapid step and toward the cytoplasmic side in the second slow step; 3) at least one electroneutral reaction precedes the electrogenic steps. Relaxation rate constant lambda3 reflects ATP binding, with lambda(3,max) approximately 100 s(-1). This step is electroneutral. Comparison with the kinetics of the reaction cycle shows that the first electrogenic step (inward current) occurs before the decay of E2P. Candidates are the formation of phosphoenzyme from E1ATP (lambda2 approximately 200 s[-1]) and the E1P --> E2P transition (D approximately 1 ms or lambda1 approximately 300 s[-1]). The second electrogenic transition (outward current) follows the formation of E2P (lambda4 approximately 3 s[-1]) and is tentatively assigned to H+ countertransport after the dissociation of Ca2+. Quenched flow experiments performed under the conditions of the electrical measurements 1) demonstrate competition by caged ATP for ATP-dependent phosphoenzyme formation and 2) yield a rate constant for phosphoenzyme formation of 200 s(-1). These results indicate that ATP and caged ATP compete for the substrate binding site, as suggested by the ATP dependence of lambda3 and favor correlation of lambda2 with phosphoenzyme formation.

This article has been cited by other articles:

				G. Bartolommei, F. Tadini-Buoninsegni, S. Hua, M. R. Moncelli, G. Inesi, and R. Guidelli Clotrimazole Inhibits the Ca2+-ATPase (SERCA) by Interfering with Ca2+ Binding and Favoring the E2 Conformation J. Biol. Chem., April 7, 2006; 281(14): 9547 - 9551. [Abstract] [Full Text] [PDF]

				F. T. Buoninsegni, G. Bartolommei, M. R. Moncelli, G. Inesi, and R. Guidelli Time-Resolved Charge Translocation by Sarcoplasmic Reticulum Ca-ATPase Measured on a Solid Supported Membrane Biophys. J., June 1, 2004; 86(6): 3671 - 3686. [Abstract] [Full Text] [PDF]

				C. Peinelt and H.-J. Apell Time-Resolved Charge Movements in the Sarcoplasmatic Reticulum Ca-ATPase Biophys. J., February 1, 2004; 86(2): 815 - 824. [Abstract] [Full Text] [PDF]

				A. Kannt, U. Pfitzner, M. Ruitenberg, P. Hellwig, B. Ludwig, W. Mantele, K. Fendler, and H. Michel Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase J. Biol. Chem., December 31, 1999; 274(53): 37974 - 37981. [Abstract] [Full Text] [PDF]