EFFECTS OF THE SUBSTITUTION OF ASN879 BY ASP ON THE pNPPase ACTIVITY OF THE Ca2+ PUMP FROM HUMAN PLASMA MEMBRANE

RINALDI DEBORA ELENA; ADAMO HUGO PEDRO

Pinamar, Pcia. Bs. As.

Congreso; XLI Reunion de la Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular (SAIB); 2005

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular (SAIB)

EFFECTS OF THE SUBSTITUTION OF ASN879 BY ASP ON THE pNPPase ACTIVITY OF THE Ca2+ PUMP FROM HUMAN PLASMA MEMBRANE Rinaldi, Débora E.; Adamo, Hugo P. IQUIFIB- Facultad de Farmacia y Bioquímica (UBA-CONICET), Buenos Aires. debirinaldi@yahoo.com.ar The Asn879 of the plasma membrane Ca2+ pump (PMCA) would be one of the  Ca2+  binding ligands. Previous studies have shown that  the substitution of Asn879 by Ala or Asp results in an enzyme devoid of Ca2+ transport activity.  We have constructed  a   mutant of the human isoform 4xb in which Asn879 was replaced by Asp. The recombinant protein was expressed in S. cerevisiae and purified by calmodulin chromatography. The mutant reconstituted in a mixture of acidic lipids was capable of hydrolyzing pNPP (p-nitrophenilphosphate) although at a very low rate compared with the wt enzyme, it was resistant to Ca2+ inhibition and the increase in the glycerol content of the reaction medium had a marginal effect on the activity of Asn879Asp. In the absence of  Ca2+, vanadate inhibited the wt  pNPPase activity with high apparent affinity while  the pNPPase activity of the  mutant remained almost unchanged up to 100 uM vanadate. As judged by the unchanged Km for pNPP the mutation did not alter directly the site of pNPP hydrolysis. Because  the phosphatase activity of the PMCA is maximal in the absence of  Ca2+ (E2-like conformation)  the efects of changing   Asn 879 by Asp are consistent with the disruption of  the Ca2+ binding site. The phosphatase activity of the Asn879Asp mutant was much lower than the expected for an enzyme stabilized in E2. These results may be accounted for if the mutation Asn879Asp blocks Ca2+ binding but retains some of the properties of the Ca2+ bound form.