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Critical effects on catalytic function produced by amino acid substitutions at Asp 804 and Asp 808 of the al isoform of Na,K‐ATPase
Author(s) -
Van Huysse James W.,
Kuntzweiler Theresa A.,
Lingrel Jerry B.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00578-9
Subject(s) - ouabain , amino acid , enzyme , biochemistry , complementary dna , microbiology and biotechnology , gene isoform , atpase , chemistry , biology , gene , sodium , organic chemistry
At two intramembrane carboxyl‐containing amino acids of the sheep al isoform of Na,K‐ATPase (Asp 804 and Asp 808 ) 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 ouabainresistant enzyme (sheep α1 RD) and the encoded enzymes were expressed in ouabain‐sensitive HeLa cells. Transfections with cDNAs carrying all Asp 804 substitutions, along with those carrying As 808 Ala, Asp 808 Asn, and Asps 808 Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp 804 and Asps 808 . Only the expression of the Asp 808 Glu enzyme produced ouabain‐resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp 804 Ala and Asp 080 Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain‐sensitive wild‐type sheep αl Na,K‐ATPase, which was expressed in ouabain‐resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity ( K d =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.