Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca2+-ATPase

Aderonke O. Adebayo, A. Enyedi, Anil K. Verma, Adelaide G. Filoteo, John T. Penniston

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [γ-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882 → Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887 → Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882..

Original languageEnglish
Pages (from-to)27812-27816
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number46
Publication statusPublished - Nov 17 1995

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Calcium-Transporting ATPases
Cell membranes
Sarcoplasmic Reticulum
Cell Membrane
Pumps
Ligands
Amino Acids
Adenosine Triphosphate
Calmodulin
Viperidae
Serine
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Adebayo, A. O., Enyedi, A., Verma, A. K., Filoteo, A. G., & Penniston, J. T. (1995). Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca2+-ATPase. Journal of Biological Chemistry, 270(46), 27812-27816.

Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca2+-ATPase. / Adebayo, Aderonke O.; Enyedi, A.; Verma, Anil K.; Filoteo, Adelaide G.; Penniston, John T.

In: Journal of Biological Chemistry, Vol. 270, No. 46, 17.11.1995, p. 27812-27816.

Research output: Contribution to journalArticle

Adebayo, AO, Enyedi, A, Verma, AK, Filoteo, AG & Penniston, JT 1995, 'Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca2+-ATPase', Journal of Biological Chemistry, vol. 270, no. 46, pp. 27812-27816.
Adebayo, Aderonke O. ; Enyedi, A. ; Verma, Anil K. ; Filoteo, Adelaide G. ; Penniston, John T. / Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca2+-ATPase. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 46. pp. 27812-27816.
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abstract = "In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [γ-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882 → Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20{\%} of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887 → Ala was expressed in greater amounts than, and had a specific activity about 50{\%} higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882..",
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