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Title: Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1more » cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.« less
Authors:
;
Publication Date:
OSTI Identifier:
6143653
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 27:22
Research Org:
Univ. of Cincinnati College of Medicine, OH (USA)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ATP-ASE; AMINO ACID SEQUENCE; HELA CELLS; MUTAGENESIS; OUABAIN; BIOLOGICAL EFFECTS; ARGININE; ASPARAGINE; ASPARTIC ACID; GENE MUTATIONS; GLUTAMINE; MAN; RATS; RECOMBINANT DNA; SENSITIVITY; SHEEP; ACID ANHYDRASES; AMIDES; AMINO ACIDS; ANIMALS; CARBOHYDRATES; CARBOXYLIC ACIDS; CARDIAC GLYCOSIDES; CARDIOTONICS; CARDIOVASCULAR AGENTS; DNA; DOMESTIC ANIMALS; DRUGS; ENZYMES; GLYCOSIDES; HYDROLASES; MAMMALS; MOLECULAR STRUCTURE; MUTATIONS; NUCLEIC ACIDS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PHOSPHOHYDROLASES; PRIMATES; RODENTS; RUMINANTS; STROPHANTHINS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques