Controlled Proteolysis of Ca2+-ATPases in Human Platelet and Non-muscle Cell Membrane Vesicles: Evidence for a Multi-sarco/Endoplasmic Reticulum Ca2+-ATPase System

T. Kovács, Elisabeth Corvazier, Béla Papp, Clarice Magnier, Raymonde Bredoux, A. Enyedi, B. Sarkadi, Jocelyne Enouf

Research output: Contribution to journalArticle

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Abstract

Two sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E∼P) formation and the immunoreactivity of the platelet Ca2+-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca2+-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4°C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca2+-ATPase antibody that had been shown to recognize a 97-kDa Ca2+-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca2+-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E∼P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37°C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.

Original languageEnglish
Pages (from-to)6177-6184
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number8
Publication statusPublished - Feb 25 1994

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Sarcoplasmic Reticulum Calcium-Transporting ATPases
Proteolysis
Calcium-Transporting ATPases
Cell membranes
Platelets
Blood Platelets
Cell Membrane
Trypsin
Antibodies
Protein Isoforms
Membrane Proteins
Cells
Digestion
Anti-Idiotypic Antibodies
Cell Line
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Controlled Proteolysis of Ca2+-ATPases in Human Platelet and Non-muscle Cell Membrane Vesicles : Evidence for a Multi-sarco/Endoplasmic Reticulum Ca2+-ATPase System. / Kovács, T.; Corvazier, Elisabeth; Papp, Béla; Magnier, Clarice; Bredoux, Raymonde; Enyedi, A.; Sarkadi, B.; Enouf, Jocelyne.

In: Journal of Biological Chemistry, Vol. 269, No. 8, 25.02.1994, p. 6177-6184.

Research output: Contribution to journalArticle

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abstract = "Two sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E∼P) formation and the immunoreactivity of the platelet Ca2+-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca2+-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4°C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca2+-ATPase antibody that had been shown to recognize a 97-kDa Ca2+-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca2+-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E∼P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37°C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.",
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AU - Corvazier, Elisabeth

AU - Papp, Béla

AU - Magnier, Clarice

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AU - Sarkadi, B.

AU - Enouf, Jocelyne

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