Localization and identification of Ca2+ATPases in highly purified human platelet plasma and intracellular membranes. Evidence that the monoclonal antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in human platelets

Abstract

The Ca2+ATPase activities of highly purified human platelet membranes prepared by high-voltage free-flow electrophoresis have been analysed by using [gamma-32P]ATP hydrolysis, recognition by antibodies and phosphoenzyme-complex formation. The Ca2+ATPase activity present in mixed membranes was found to be predominantly associated with intracellular membranes after subfractionation, with only a low level of activity associated with plasma membranes. The intracellular-membrane Ca2+ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca2+ATPase was not significantly affected, suggesting that the latter does not belong to the SERCA (sarco-endoplasmic-reticulum Ca2+ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca2+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 corresponding bands in red-blood-cell membranes, confirming the Ca2+ATPase to be of the PMCA (plasma-membrane Ca2+ATPase) type. No recognition of any band was detected in intracellular membranes. Identification of the intracellular-membrane Ca2+ATPase activity was carried out 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 recognized the 100 kDa Ca2+ATPase in mixed membranes and intracellular membranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and antiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca2+ATPase recognized by PL/IM 430 was immunoprecipitated, and the immunoprecipitated protein was specifically recognized by the antiserum N89, but not by S.2b. Analysis of the phosphoenzyme-complex formation revealed potent phosphorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 430 and N89. These studies report the presence of a PMCA in a purified plasma-membrane fraction from human platelets, and that the antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in intracellular membranes.

Thapsigargin discriminates strongly between Ca2+‐ATPase phosphorylated intermediates with different subcellular distributions in bovine adrenal chromaffin cells

“…However, additional structurally distinct family members appear to exist [4], suggesting that the full extent of the heterogeneity among SERCA pumps has not yet been explored. The characteristic tissue distribution [3], as well as the occurrence of multiple SERCA isoenzymes in one cell type [5][6][7] suggest functional implications *Corresponding author. Department of Medical Physiology, The Panum Institute, Blegdamsvej 3C, 2200 N, Copenhagen, Denmark.…”

Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel

“…Fluorescence measurements were made using a rotating wheel spectrofluorimeter (Cairns Research, Faversham Preparation of highly purified platelet plasma and intracellular membranes using high-voltage free flow electrophoresis Platelet plasma membranes (PMs) and intracellular membranes (IMs) were prepared as described in considerable detail in previous publications. 32,33 Briefly, platelets were separated from human blood and treated with neuraminidase (type X, 0.05 U/mL) for 20 minutes at 37°C. After washing, platelets were sonicated at 4°C (with 2 ϫ 10-second bursts, amplitude setting at 50) in sonication medium and centrifuged at 42 000g for 90 minutes on a linear (1-3.5 M) sorbitol density gradient to obtain a mixed membrane (MM) fraction (free of granular contamination).…”

All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells

“…Although the PL\IM 430 monoclonal antibody has been reported to immunostain a 97 kDa SERCA protein in various human non-muscle cells [19,20], the identity of this SERCA variant has long been debated [21][22][23]. Poch and his co-workers [28] provided evidence that this antibody recognizes both the cloned human SERCA3a and 3c isoforms expressed in HEK-293 SERCA3a, 3b and 3c protein isoforms in haematopoietic cells cells.…”

“…In human non-muscle tissues the housekeeping SERCA2b [15,16] and a SERCA3 isoform [17,18] (SERCA3a according to the recent nomenclature) have been identified. The presence of a SERCA protein recognized by PL\IM 430, a monoclonal anti-SERCA antibody raised against highly purified platelet intracellular membranes, has also been demonstrated [19,20] ; however, the identity of this SERCA form has long been debated [21][22][23]. While two alternatively spliced variants have long been known for both SERCA1 and SERCA2 [14][15][16]24,25], alternative splicing of the human SERCA3 pre-mRNA has only recently been reported by three independent groups [26][27][28].…”