Dissection of the functional differences between human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and transient kinetic analyses.
Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.
Dode
Leonard
L
Laboratory of Physiology, Catholic University of Leuven, Campus Gasthuisberg O/N, Herestraat 49, Bus 802, B-3000 Leuven, Belgium.
Andersen
Jens Peter
JP
Vanoevelen
Jo
J
Raeymaekers
Luc
L
Missiaen
Ludwig
L
Vilsen
Bente
B
Wuytack
Frank
F
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D020033:Protein Isoforms; D013696:Temperature; D019284:Thapsigargin / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D007476:Ionophores; D020033:Protein Isoforms; D019284:Thapsigargin; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D011188:Potassium; D002118:Calcium