High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.
The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.
Cura
Carolina I
CI
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB)-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Ciudad de Buenos Aires, Argentina. carocura@hotmail.com
Corradi
Gerardo R
GR
Rinaldi
Débora E
DE
Adamo
Hugo P
HP
D000143:Acids / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D050356:Lipid Metabolism; D008823:Micelles; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology
D000143:Acids; D002147:Calmodulin; D003902:Detergents; D007527:Isoenzymes; D008823:Micelles; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D000255:Adenosine Triphosphate; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases