Abstract
The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca(2+) transport from ATP hydrolysis in platelet membranes (Hack, N., Wilkinson, J. M., and Crawford, N. (1988) Biochem. J. 250, 355-361). More recently, we have demonstrated that this antibody is specific for human SERCA3 (Poch, E., Leach, S., Snape, S., Cacic, T., MacLennan, D. H., and Lytton, J. (1998) Am. J. Physiol. 275, C1449-C1458). In this paper, we have extended the analysis of the PL/IM430-SERCA3 interaction. Using HEK293 cells to express human SERCA3a, we were able to measure both ATP-mediated, oxalate-dependent (45)Ca(2+) uptake and Ca(2+)-dependent ATP hydrolysis activities due exclusively to SERCA3. Treatment with PL/IM430 inhibited both activities almost identically, with a maximal inhibition of 81 and 73% and a half-maximal concentration of 8.3 and 5.9 microg/ml, for Ca(2+) uptake and ATP hydrolysis, respectively. We conclude that PL/IM430 does inhibit SERCA3 activity but does not uncouple Ca(2+) transport from ATP hydrolysis. Using a combination of partial proteolysis, GST fusion protein expression, and mutation of residues that differ between rat and human SERCA3, we have identified human SERCA3 amino acids Pro(8) and Glu(192) as essential to forming the PL/IM430 epitope. PL/IM430 thus recognizes a linearly noncontiguous set of amino acids within the actuator domain of human SERCA3. We propose that PL/IM430 inhibits SERCA3 activity by sterically preventing movement of the actuator domain into a catalytically critical position in the E2 conformation of the enzyme.
Highlights
The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca2؉ transport from ATP hydrolysis in platelet membranes
Antibodies developed against intracellular platelet memformational states of the SERCA enzyme, which is stabilized by the binding of the right-hand column (Tg), as determined by Toyoshima et al [28]; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.1% Tween 20; SERCA, sarcoplasmic or endoplasmic reticulum Ca2ϩATPase; SR, sarcoplasmic reticulum; MOPS, 4-morpholinepropanesulfonic acid
The HEK293 microsome system allows the measurement of SERCA activity corresponding unambiguously to the protein encoded by the transfected cDNA
Summary
Probing the PL/IM430 –SERCA3 Interaction branes have been used widely as a tool to study the multiSERCA system in platelets One such antibody is PL/IM430 (platelet intracellular membrane 430), which recognizes all alternatively spliced SERCA3 isoforms [9]. This antibody was reported to inhibit Ca2ϩ uptake into, but not ATP hydrolysis by, highly purified platelet intracellular membranes, suggesting that PL/IM430 was able to uncouple Ca2ϩ transport from ATP hydrolysis in the SERCA3 protein [16] This observation, if confirmed, would be of fundamental importance for the further understanding of structure-function relationships involved in conformational coupling accompanying active ion transport. We have identified the epitope for this inhibitory antibody These data have important implications in understanding the conformational changes accompanying active Ca2ϩ transport mediated by the SERCA pumps
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