“Substitution of Aspartate for Asparagine 879 in the human plasma membrane Ca2+ pump”

DEBORA E RINALDI; HUGO P. ADAMO

UBA. Buenos Aires. Argentina

Simposio; Simposio Internacional de la Maestría Internacional en Ciencias Biomédicas de la Universidad de Buenos Aires y de la Universidad Albert Ludwigs de Friburgo (Alemania); 2008

.O {font-size:149%;} <!--.sld {left:0px !important; width:6.0in !important; height:8.0in !important; font-size:138% !important;} --> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0pt; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-AR;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 85.05pt 70.85pt 85.05pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The reaction cycle of the plasma membrane Ca2+ pump is normally described using the E1E2 model in which the catalytic specifitity of the enzyme is governed by Ca2+. The maximal ATP hydrolytic activity (Ca-ATPase) is asociated with the E1 conformer which is stabilized by Ca2+. On the other hand the E2 form of the enzyme is able to hydrolyse p-nitrophenilphosphate (pNPP) in the absence of Ca2+. A mutant of the human isoform 4xb in wich Asn879 was replaced by Asp was expressed in Saccharomyces cerevisiae, solubilized by the detergent C12E10 and purified by calmodulin chromatography. In agreement with the previously  proposed esential role in the formation of a high affinity site for Ca2+, the Ca-ATPase activity of the Asn879Asp mutant was 12% of that of the wild type enzyme. Most strikingly it exhibited a   Ca2+ dependency similar to the wild type. Furthermore the mutant reconstituted in a mixture of acidic lipids was capable of hydrolyzing pNPP in the absence of Ca2+ although the p-nitrophenilphosphatase activity was about 15% of that of the wild type enzyme and it was resistant to Ca2+ inhibition. These result suggest that the effects of the mutation are much more complex than expected and involve alterations in the catalytic activity of the enzyme both in the absence and in the presence of Ca2+. With grants from UBACyT, CONICET and ANPCyT.