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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

Abstract

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:
Research Org.:
Univ. of Cincinnati College of Medicine, OH (USA)
OSTI Identifier:
6143653
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 27:22
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

Citation Formats

Price, E.M., and Lingrel, J.B. 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. United States: N. p., 1988. Web.
Price, E.M., & Lingrel, J.B. 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. United States.
Price, E.M., and Lingrel, J.B. 1988. "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". United States. doi:.
@article{osti_6143653,
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},
author = {Price, E.M. and Lingrel, J.B.},
abstractNote = {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..1 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.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 27:22,
place = {United States},
year = 1988,
month =
}
  • Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the 'aspartate-rich' motif D100DXX(D/E) that coordinates to Mg{sup 2+}{sub A} and Mg{sup 2+}{sub C} source, and the 'NSE/DTE' motif N225DXXSXXXE that chelates Mg{sup 2+}{sub b} (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpenemore » products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.« less
  • Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using (1{beta},2{beta}-{sup 3}H)androstenedione, (19-{sup 14}C)androstenedione, and (1{beta},2{beta}-{sup 3}H)testosterone as substrates. The mutant Phe-406{r arrow} Arg was completely inactive. Since there were only small changes in the K{sub m} and V{sub max} values for all substrates for mutants Tyr-3561{r arrow} Phe and Tyr-361{r arrow} Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308{rmore » arrow} Phe had altered catalytic properties; the K{sub m} values for androstenedione, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.« less
  • Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 ..-->.. Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilidemore » (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 ..-->.. Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with ..-->../sub 2/-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form.« less
  • X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of asparate aminotransferase. It forms a salt bridge with the ..beta.. or ..gamma.. carboxylate group of the substrate. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity k/sub cat//K/sub M/) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and ..cap alpha..-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate andmore » a number of aromatic and other neutral amino acids is reduced by only 2-9-fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (k/sub cat//K/sub M/)/sub cationic//(k/sub cat//K/sub M/)/sub anionic/ is in the range 2-40-fold for R292D, whereas this ratio has a range of ((0.3-6) x 10/sup -6/)-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity. Possible explanations for the less-than-expected reactivity of R292D with arginine are discussed.« less