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

• Autorzy: Sobczak A. , Blazejczyk M. , Piszczek G. , Zhao G. , Kuznicki J. , Wojda U.
• Języki publikacji: EN

Abstrakty

EN

The EF-hand Ca2+-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca2+ signals. As oligomerization may represent one of Ca2+-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 Ca2+-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 Ca2+-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 Ca2+. 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 Ca2+ concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.

Słowa kluczowe

EN

calmyrin monomer; calcium-binding calmyrin form; man; lymphocyte; human lymphocyte; in vivo; covalent dimer; in vitro; monomeric form

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2005
• Tom: 52
• Numer: 2
• Opis fizyczny: p.469-476,fig.,ref.

Twórcy

autor

Sobczak A.

• International Institute of Molecular and Cell Biology, Warsaw, Poland

autor

Blazejczyk M.

autor

Piszczek G.

autor

Zhao G.

autor

Kuznicki J.

autor

Wojda U.

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