Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form
Sobczak, A.; Blazejczyk, M.; Piszczek, G.; Zhao, G.; Kuznicki, J.; Wojda, U.
Acta Biochimica Polonica 52(2): 469-476
2005
ISSN/ISBN: 0001-527X PMID: 15933764 Document Number: 585003
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 mu M 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 K-a = 1.78 x 10(3) M-1 at 6 degrees C. Gel filtration of apo- and Ca2+-bound calmyrin at a 100 mu M 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 anal lyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected inhuman 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.