{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T23:13:21Z","timestamp":1791328401690,"version":"4.3.1"},"reference-count":52,"publisher":"American Physiological Society","issue":"5","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2009,5]]},"abstract":"<jats:p> The sarco(endo)plasmic reticulum Ca<jats:sup>2+<\/jats:sup>-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca<jats:sup>2+<\/jats:sup> concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca<jats:sup>2+<\/jats:sup>; however, Ca<jats:sup>2+<\/jats:sup> affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca<jats:sup>2+<\/jats:sup> uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca<jats:sup>2+<\/jats:sup> activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL\/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca<jats:sup>2+<\/jats:sup> affinity for SERCA3 compared with SERCA2b (1.10 \u00b1 0.04 vs. 0.26 \u00b1 0.01 \u03bcM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca<jats:sup>2+<\/jats:sup> affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca<jats:sup>2+<\/jats:sup> affinity in these two preparations was 1.04 \u00b1 0.07 and 1.1 \u00b1 0.2 \u03bcM for SERCA3 and 0.27 \u00b1 0.02 and 0.26 \u00b1 0.01 \u03bcM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca<jats:sup>2+<\/jats:sup> is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms. <\/jats:p>","DOI":"10.1152\/ajpcell.00650.2008","type":"journal-article","created":{"date-parts":[[2009,2,19]],"date-time":"2009-02-19T02:54:10Z","timestamp":1235012050000},"page":"C1105-C1114","source":"Crossref","is-referenced-by-count":48,"title":["Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3"],"prefix":"10.1152","volume":"296","author":[{"given":"P. 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