Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets

T. Kovács, Gaetan Berger, Elisabeth Corvazier, K. Pászty, Angie Brown, Régis Bobe, Béla Papp, Frank Wuytack, Elisabeth M. Cramer, Jocelyne Enouf

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Abstract

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

Original languageEnglish
Pages (from-to)192-203
Number of pages12
JournalBritish Journal of Haematology
Volume97
Issue number1
Publication statusPublished - 1997

Fingerprint

Sarcoplasmic Reticulum Calcium-Transporting ATPases
Protein Isoforms
Blood Platelets
Intracellular Membranes
Membranes
Cell Membrane
Antibodies
Western Blotting
Subcellular Fractions
Immunoelectron Microscopy
Calcium-Transporting ATPases
Neuraminidase
Membrane Potentials
Flow Cytometry
Monoclonal Antibodies

Keywords

  • Ca
  • Ca pools
  • immunolocalization
  • platelets
  • SERCA isoforms

ASJC Scopus subject areas

  • Hematology

Cite this

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets. / Kovács, T.; Berger, Gaetan; Corvazier, Elisabeth; Pászty, K.; Brown, Angie; Bobe, Régis; Papp, Béla; Wuytack, Frank; Cramer, Elisabeth M.; Enouf, Jocelyne.

In: British Journal of Haematology, Vol. 97, No. 1, 1997, p. 192-203.

Research output: Contribution to journalArticle

Kovács, T, Berger, G, Corvazier, E, Pászty, K, Brown, A, Bobe, R, Papp, B, Wuytack, F, Cramer, EM & Enouf, J 1997, 'Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets', British Journal of Haematology, vol. 97, no. 1, pp. 192-203.
Kovács, T. ; Berger, Gaetan ; Corvazier, Elisabeth ; Pászty, K. ; Brown, Angie ; Bobe, Régis ; Papp, Béla ; Wuytack, Frank ; Cramer, Elisabeth M. ; Enouf, Jocelyne. / Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets. In: British Journal of Haematology. 1997 ; Vol. 97, No. 1. pp. 192-203.
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AU - Brown, Angie

AU - Bobe, Régis

AU - Papp, Béla

AU - Wuytack, Frank

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