LOCALIZATION AND IDENTIFICATION OF CA(2- EVIDENCE THAT THE MONOCLONAL-ANTIBODY PL()ATPASES IN HIGHLY PURIFIED HUMAN PLATELET PLASMA AND INTRACELLULAR MEMBRANES )IM-430 RECOGNIZES THE SERCA-3 CA(2+)ATPASE IN HUMAN PLATELETS/

The Ca(2+)ATPase activities of highly purified human platelet membrane s prepared by high-voltage free-flow electrophoresis have been analyse d by using [gamma-P-32]ATP hydrolysis, recognition by antibodies and p hosphoenzyme-complex formation. The Ca(2+)ATPase activity present in m ixed membranes was found to be predominantly associated with intracell ular membranes after subfractionation, with only a low level of activi ty associated with plasma membranes. The intracellular-membrane Ca(2+) ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca(2+)ATPase was not significantly affected, sugge sting that the latter does not belong to the SERCA (sarco-endoplasmic- reticulum Ca(2+)ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca(2+)ATPase [Cheng, Magocsi, Cooper, Penniston and Borke (1993) Cell Physiol. Biochem. 4, 31-43] recognized two bands at 135 and 150 kDa in mixed membranes and plasma membranes, and the c orresponding bands in red-blood-cell membranes, confirming the Ca(2+)A TPase to be of the PMCA (plasma-membrane Ca(2+)ATPase) type. No recogn ition of any band was detected in intracellular membranes. Identificat ion of the intracellular-membrane Ca(2+)ATPase activity was carried ou t with polyclonal antibodies with known specificity towards SERCA 2b ( S.2b) and SERCA 3 (N89), and a monoclonal antibody, PL/IM 430, raised against platelet intracellular membranes. All of these antibodies reco gnized the 100 kDa Ca(2+)ATPase in mixed membranes and intracellular m embranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and an tiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca(2+)ATPase recogniz ed by PL/IM 430 was immunoprecipitated, and the immunoprecipitated pro tein was specifically recognized by the antiserum N89, but not by S.2b . Analysis of the phosphoenzyme-complex formation revealed potent phos phorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 4 30 and N89. These studies report the presence of a PMCA in a purified plasmamembrane fraction from human platelets, and that the antibody PL /IM 430 recognizes the SERCA 3 Ca(2+)ATPase in intracellular membranes .