Functional domains of sarcoplasmic reticulum Ca2+-ATPase studied by site-directed mutagenesis

摘要: The availability of the cDNA clones P-type ATPases has opened up possibility for introducing defined point mutations in proteins by vitro oligonucleotide-directed mutagenesis cDNA. This approach can be used to search residues involved ligand binding or critical conformational transitions, provided a suitable system is available functional expression mutant In addition requirement high level exogenous cDNA, absence significant contribution from endogenous pumps demanded. sarcoplasmic reticulum Ca2+-ATPase been succesfully expressed COS-1 cells, at levels more than 100-fold higher cell Ca2+-pump. permitted analysis 200 different mutants (2–11,16–17,19–22,24). general strategy assay first ATP-driven active Ca2+ uptake and ATPase activity isolated microsomal fraction containing enzyme, thereafter phosphorylation ATP Pi, its substrate dependence, as well dephosphorylation kinetics presence ADP. Recently, it proved possible measure Ca2+-occlusion (24).