Functional comparison between secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses.
Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca(2+) transport cycle of the human secretory pathway Ca(2+)/Mn(2+)-ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200-350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca(2+) in activation of the overall ATPase and phosphorylation activities. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E(1) approximately P(Ca) to E(2)-P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E(2)-P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K(+).
Dode
Leonard
L
Laboratory of Physiology, Catholic University of Leuven, Campus Gasthuisberg O/N, Herestraat 49, Bus 802, B-3000 Leuven, Belgium. leonard.dode@med.kuleuven.be
Andersen
Jens Peter
JP
Raeymaekers
Luc
L
Missiaen
Ludwig
L
Vilsen
Bente
B
Wuytack
Frank
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D014638:Vanadates; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium