Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles

Béla Papp, Agnes Enyedi, Tünde Kovács, Balázs Sarkadi, Frank Wuytack, Ole Thastrup, George Gárdos, Raymonde Bredoux, Sylviane Levy-Toledano, Jocelyne Enouf

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Abstract

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.

Original languageEnglish
Pages (from-to)14593-14596
Number of pages4
JournalJournal of Biological Chemistry
Volume266
Issue number22
Publication statusPublished - 1991

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Thapsigargin
Platelets
Demonstrations
Blood Platelets
Pumps
Calcium
Membranes
Protein Isoforms
Calcium-Transporting ATPases
Endoplasmic Reticulum
Immune Sera
Adenosine Triphosphatases
Lanthanum
Active Biological Transport
Sarcoplasmic Reticulum
Enzymes
Autoradiography
Immunoblotting
Membrane Proteins
Molecular mass

ASJC Scopus subject areas

  • Biochemistry

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Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. / Papp, Béla; Enyedi, Agnes; Kovács, Tünde; Sarkadi, Balázs; Wuytack, Frank; Thastrup, Ole; Gárdos, George; Bredoux, Raymonde; Levy-Toledano, Sylviane; Enouf, Jocelyne.

In: Journal of Biological Chemistry, Vol. 266, No. 22, 1991, p. 14593-14596.

Research output: Contribution to journalArticle

Papp, B, Enyedi, A, Kovács, T, Sarkadi, B, Wuytack, F, Thastrup, O, Gárdos, G, Bredoux, R, Levy-Toledano, S & Enouf, J 1991, 'Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles', Journal of Biological Chemistry, vol. 266, no. 22, pp. 14593-14596.
Papp, Béla ; Enyedi, Agnes ; Kovács, Tünde ; Sarkadi, Balázs ; Wuytack, Frank ; Thastrup, Ole ; Gárdos, George ; Bredoux, Raymonde ; Levy-Toledano, Sylviane ; Enouf, Jocelyne. / Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 22. pp. 14593-14596.
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AU - Enyedi, Agnes

AU - Kovács, Tünde

AU - Sarkadi, Balázs

AU - Wuytack, Frank

AU - Thastrup, Ole

AU - Gárdos, George

AU - Bredoux, Raymonde

AU - Levy-Toledano, Sylviane

AU - Enouf, Jocelyne

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AB - In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.

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