CRITICAL EFFECTS ON CATALYTIC FUNCTION PRODUCED BY AMINO-ACID SUBSTITUTIONS AT ASP(804) AND ASP(808) OF THE ALPHA-1 ISOFORM OF NA,K-ATPASE

At two intramembrane carboxyl-containing amino acids of the sheep alph a 1 isoform of Na,K-ATPase (Asp(804) and Asp(808)), both charge-conser ving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replac ements were made and the altered enzymes studied, Nucleotide changes e ncoding the amino acid substitutions were placed in a cDNA encoding a ouabain-resistant enzyme (sheep alpha 1 RD) and the encoded enzymes we re expressed in ouabain-sensitive HeLa cells, Transfections with cDNAs carrying all Asp(804) substitutions, along with those carrying Asp(80 8)Ala, Asp(808)Asn, and Asp(808)Leu replacements failed to confer ouab ain resistance to the cells, indicating critical roles for Asp(804) an d Asp(808). Only the expression of the Asp(808)Glu enzyme produced oua bain-resistant HeLa cells, demonstrating that the altered protein was functional, When the inactive proteins Asp(804)Ala and Asp(808)Ala wer e expressed using an alternative selection system (the protein carryin g the amino acid substitution was the ouabain-sensitive wild-type shee p alpha 1 Na,K-ATPase, which was expressed in ouabain-resistant 3T3 ce lls), intact cells were able to bind extracellular ouabain with high a ffinity (K-d = 1-30 nM), indicating that the inactive proteins were sy nthesized and folded properly in the plasma membrane, The results demo nstrate that carboxyl side chains at positions 804 and 808 are critica l for enzyme catalytic function.