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A lipid binding protein is involved in the Mg.ATP stimulation of squid Na+/Ca2+ exchange
BERBERIAN, P., BOLLO, M., ROBERTS G., DEGIORGIS, J., DIPOLO R. AND BEAUGE L.
Congreso; 6th International Conference on Biological Physics (ICBP) and 5th Southern Cone Biophysics Congress; 2007
ICBP 2007 - Abstract Submission Paper Nº: 145 15-Membranes and Transport Presenter: Berberian, G First Author: Berberian, G (Argentina) Registered: Berberian, G Contact:Berberian, Graciela(Argentina)email@example.com A lipid binding protein is involved in the Mg.ATP stimulation of squid Na/Ca exchange.15-Membranes and Transport Berberian, G1; Bollo, M2; Roberts, G2; DeGiorgis, J3; DiPolo, R4; Beaugé, L51 - Instituto M. y M. Ferreyra- INIMEC- CONICET, Argentina; Marine Biological Laboratory, Woods Hole, USA. 2 - Instituto M. y M. Ferreyra- INIMEC- CONICET, Argentina. 3 - Marine Biological Laboratory, Woods Hole, USA. 4 - Laboratorio de Fisilogía Celular, IVIC, Venezuela ; Marine Biological Laboratory, Woods Hole, USA. 5 - Instituto M. y M. Ferreyra- INIMEC- CONICET, Argentina;Marine Biological Laboratory, Woods Hole, USA. Abstract: We have previously demonstrated that up-regulation by MgATP of the squid Na+/Ca2+ exchanger (dialyzed giant axons or membrane vesicles from optic nerve) requires a soluble cytosolic protein of about 13 kDa (SCRP). We have also shown that, even in the absence of MgATP, this protein can stimulate the exchanger when is previously phosphorylated by MgATP by kinase/s present in the plasma membrane of the squid nerve (Ann NY Acad. Sci. 1099,152-165, 2007). Here we provide preliminary evidence on the identification of this SCRP. A doublet of cytosolic phosphorylated bands of about 13 kDa were purified from a gradient SDS-PAGE and the amino acids of both bands were sequenced (W.M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia Biomedical Research Facility). They were identified as belonging to the family of lipid binding protein. These two proteins were cloned and expressed in E. coli. The two clones (clone 76 and clone 68) were purified by affinity chromatography and assayed for their biological activities. Clone 76 became phosphorylated by Mg.ATP in the presence of squid nerve membranes and was able to promote MgATP stimulation of Na+/Ca2+ exchange fluxes in squid nerve vesicles. On the other hand, in microsomes obtained from squid optic ganglia, Na+/Ca2+ exchange could be stimulated by MgATP even in the absence of any cytosolic fraction. As a difference with membranes vesicles from squid optic nerve, in these microsomes a protein of about 13kDa is present in the membrane and becomes phosphorylated by MgATP. Both the MgATP stimulation of the exchanger and the phosphorylation of the 13 kDa proteins are insensitive to Staurosporine. In addition, opposite to what happens in the bovine sarcolemmal, PtdIns-P2 does not affect the squid nerve Na+/Ca2+ exchanger. Supported by Grants from the US National Science Found ation (MCB 0444598), FONCYT (PICT-05-12397 and PICT-05-38073) and CONICET (PIP 5118), Argentina, and Fonacit (S1-9900009046 and G-2001000637) and Fundación Polar, Venezuela.