CONSTRUCTION OF AN AUTOFLUORESCENT PLASMA MEMBRANE CA2+ PUMP.

GERARDO R CORRADI; HUGO P ADAMO

Buenos Aires Argentina

Congreso; Iupab. InternationalBiophysics Congress; 2002

The plasma membrane Ca2± pump (PMCA) is a calmodulinregulated P-type ATPase that is essential for controlling intracellular Ca2+ concentration. Using the megaprimer method for mutagenesis, we have constructed a DNA coding for a the human PMCA (isoform 4bx) with the SuperGloGFP (green fluorescent protein, Quantum biogen, CA, USA) fused to its the C-terminal end. The mutant DNA was inserted in the vector pYX-TEV for expression in Saccharomyces cerevisiae. Immunoblots using antiPMCA antibodies showed that tOe PMCA-GFP was correctly expressed in the yeast mernbranes. The PMCA-GFP was purified by calmodulin-affinity chromatography asad reconstituted into liposomes of phosphatidylcholine. Like the wild type GFP, the purified PMCA-GFP exhibited a maximal emission at 509 nm when excited at 474 nm. ha the presence of Ca2+ the PMCA-GFP was able of hydrolyzing ATP at a rate of 50 nmollmg proteinlmin. a value close lo that of the recombinant wild type PMCA. The addition of 240 nM calmodulin to thc reaction medium increased 3-4 fold the activity of the PMCA-GFP. These results indicate that thc PMCA-GFP is a fully active, calmodulin regulated PMCA that can be directly detected by its autofluorescence. Supported by the ANPCyT PtCT 1-6845. POSTO4-25