“Some biochemical properties of the up-regulation by MgATP and phosphoarginine of the squid nerve Na+/Ca2+ exchanger”.

ELSO DE BERBERIÁN, G.; DIPOLO, R., AND L. BEAUGÉ

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES.

N.Y. Acad. Sci

Lugar: Los Angeles; California, USA; Año: 2006 vol. 1099

0077-8923

SOME BIOCHEMICAL PROPERTIES OF THE UP-REGULATION BY MgATP AND PHOSPHOARGININE OF THE SQUID NERVE Na+/Ca2+ EXCHANGER                                                     by          Graciela Berberiána,c, Reinaldo DiPolob,c and Luis Beaugéa,c    a Laboratorio de Biofísica, Instituto de Investigación Médica “Mercedes y Martín Ferreyra” (INIMEC-CONICET), Casilla de Correo 389, 5000 Córdoba, Argentina b Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, IVIC, Apartado 21827, Caracas1020-A, Venezuela c Marine Biological Laboratory, Woods Hole, USA        ABSTRACT In squid nerve MgATP up-regulation of Na+/Ca2+ exchange requires a soluble cytosolic regulatory protein (SCRP) of about 13 kD; PA stimulation does not. MgATP-γ-S mimics MgATP. When the 30-10 kD cytosolic fraction is exposed to 0.5 mM [32P]ATP in the same solution used for transport assays, and in the presence of native membrane vesicles, a 13 kD and a 25 kD band become phosphorylated. Membrane vesicles alone do not show these two phosphorylated bands and heat denaturation of the cytosolic fraction prevents phosphorylation. Moreover, Staurosporine, a general inhibitor of kinases, does not affect MgATP + SCRP stimulation of the exchanger or the phosphorylation of the 13 kD cytosolic band but prevents phosphorylation of the 25 kD band. The phosphorylated 30-10 kD fraction can stimulate the Na+/Ca2+ exchanger in vesicles even in the absence of ATP provided there is Mg2+ in the media. The phosphorylation pattern of membrane proteins by PA is different from that of MgATP: two bands phosphorylated by PA, at about 60 kD and 70 kD, not seen with MgATP, correspond to the low molecular weight neurofilament (NF). The PA-phosphorylated NF is specific for PA, insensitive to Staurosporine (similar to the PA stimulated fluxes) and labile, loosing the [32P]Pi upon immunoprecipitation. In addition, co-immunoprecipitation was observed between the NF  and the exchanger protein. Under the conditions of these experiments no phosphorylation of the exchanger is detected, either with ATP or PA .