Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the alpha1 isoform of Na,K-ATPase.
At two intramembrane carboxyl-containing amino acids of the sheep alpha1 isoform of Na,K-ATPase (Asp804 and Asp808), both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabain-resistant enzyme (sheep alpha1 RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all Asp804 substitutions, along with those carrying Asp808Ala, Asp808Asn, and Asp808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp804 and Asp808. Only the expression of the Asp808Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp804Ala and Asp808Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep alpha1 Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (Kd = 1-30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.
Van Huysse
J W
JW
Department of Molecular Genetics, University of Cincinnati College of Medicine, OH 45267-0524, USA.
Kuntzweiler
T A
TA
Lingrel
J B
JB
D016475:3T3 Cells / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D004789:Enzyme Activation; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase