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2005 | 52 | 2 | 469-476
Article title

Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.

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EN
Abstracts
EN
The EF-hand Ca^(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca^(2+) signals. As oligomerization may represent one of Ca^(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca^(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 µM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 × 103 M^(-1) at 6oC. Gel filtration of apo- and Ca^(2+)-bound calmyrin at a 100 µM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca^(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca^(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.
Publisher

Year
Volume
52
Issue
2
Pages
469-476
Physical description
Dates
published
2005
received
2005-02-10
revised
2005-02-25
accepted
2005-04-19
(unknown)
2005-06-01
Contributors
author
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Laboratory of Biochemistry, NHLBI/NIH, Bethesda, MD 20892-8012, USA
author
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Nencki Institute of Experimental Biology, Laboratory of Calcium-Binding Proteins, Warsaw, Poland
author
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
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Document Type
Publication order reference
Identifiers
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv52i2p469kz
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