Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the al isoform of Na,K‐ATPase

FEBS Letters

Kuntzweiler, Theresa A.¹  Lingrel, Jerry B.¹  Van Huysse, James W.¹ 

[1] Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0524, USA

关键词: Na;    K-ATPase;    Site-directed mutagenesis;    Cation dependence;    Ouabain;    Protein processing;   

DOI  :  10.1016/0014-5793(96)00578-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

At two intramembrane carboxyl-containing amino acids of the sheep al isoform of Na,K-ATPase (Asp ⁸⁰⁴ 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 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 As⁸⁰⁸Ala, Asp⁸⁰⁸ Asn, and Asps⁸⁰⁸Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp⁸⁰⁴ and Asps⁸⁰⁸. Only the expression of the Asp ⁸⁰⁸Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp⁸⁰⁴Ala and Asp⁰⁸⁰Ala 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.

【 授权许可】

Unknown