Dissection of the Functional Differences between Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) 1 and 3 Isoforms by Steady-state and Transient Kinetic Analyses

Leonard Dode, Bente Vilsen, Kurt Van Baelen, Frank Wuytack, Johannes D. Clausen, Jens Peter Andersen
2002 Journal of Biological Chemistry  
Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca 2؉ -transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca 2؉ in activation of the overall reaction due to a decreased E 2 to E 1 Ca 2 transition rate and an increased rate of Ca 2؉ dissociation
more » ... a 2؉ dissociation from E 1 Ca 2 . At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca 2؉ level (either an enhanced efflux of lumenal Ca 2؉ through the pump in E 2 form or insensitivity to inhibition by lumenal Ca 2؉ ). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca 2؉ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E 2 P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus. . 1 The abbreviations used are: SERCA, sarco(endo)plasmic reticulum Ca 2ϩ -ATPase; E 1 , enzyme form with cytoplasmically facing high-affinity Ca 2ϩ sites; E 2 , enzyme form with low affinity for Ca 2ϩ ; E 1 ϳP(Ca 2 ), phosphoenzyme with high-energy phosphoryl group (transferable to ADP) and occluded calcium ions (shown in parentheses); E 2 P, phosphoenzyme with low-energy phosphoryl group and lumenally facing low-affinity Ca 2ϩ sites; M1-M10, the transmembrane segments numbered from the N terminus of the Ca 2ϩ -ATPase; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; S5, the fifth stalk segment; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.
doi:10.1074/jbc.m207778200 pmid:12207029 fatcat:igglxttjgvhlhkv2zy6ajvum6q