Expression, purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity.
The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.
Guerini
Danilo
D
Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8092 Zürich, Switzerland.
Pan
Bin
B
Carafoli
Ernesto
E
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D027682:Cation Transport Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases; D011485:Protein Binding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
C506952:ATP2B1 protein, human; D002147:Calmodulin; D027682:Cation Transport Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014357:Trypsin; D002154:Calpain; D000251:Adenosine Triphosphatases; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium