Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…The resulting model showed important roles for several of the amino acids that were identified in both studies with the chimeras [13,14]. In addition, the two amino acids discovered by Lingrel's group [15] were very important in the model.…”

“…The chimeras and mutants used in this study were: the rat Na,K-ATPase α 1 -subunit (wild type), the R111Q/ D122N mutant (QN) of this enzyme [10,11], the rat non-gastric H, K-ATPase α 2 -subunit [13] as well as its EGPLC mutant (D312E, S319G, A778P, I795L, F802C) [14]. Generation of the vectors containing the α 2 -subunit of rat non-gastric H,K-ATPase with the β 1 -subunit of rat Na,K-ATPase or the α 1 -subunit of rat Na,K-ATPase with the β 1 -subunit of sheep Na,K-ATPase that were suited for the baculovirus expression system, has been reported before [5,14].…”

“…Generation of the vectors containing the α 2 -subunit of rat non-gastric H,K-ATPase with the β 1 -subunit of rat Na,K-ATPase or the α 1 -subunit of rat Na,K-ATPase with the β 1 -subunit of sheep Na,K-ATPase that were suited for the baculovirus expression system, has been reported before [5,14].…”

“…In a next study, we succeeded to convert the rat nongastric H,K-ATPase that has a very low affinity for ouabain, by mutation of only five amino acids into an enzyme with a high affinity for ouabain [14]. Two of these amino acids (Glu 312 and Gly 319 ) were also found to be important in the study with the gastric H,K-ATPase chimera EVIGFTD, whereas three others Pro 778 , Leu 795 and Cys 802 were new.…”

“…Two of these amino acids (Glu 312 and Gly 319 ) were also found to be important in the study with the gastric H,K-ATPase chimera EVIGFTD, whereas three others Pro 778 , Leu 795 and Cys 802 were new. This chimera (EGPLC) could, in contrast to chimera EVIGFTD, well be expressed in the baculovirus system and showed a high NH þ 4 -activated ATPase activity that could be inhibited by low ouabain concentrations [14].…”