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1.
Biochemistry ; 36(4): 941-51, 1997 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-9020794

RESUMO

A model for the active site of P type ATPases has been tested by site-directed mutagenesis of amino acids in two conserved sequences of Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase. The mutants K501R, K501E, D586E, D586N, P587A, and P588A were expressed in yeast cells and compared with wild type. In addition to previously published assays of adenosine 5'-triphosphate binding and hydrolysis, measurements of 18O exchange between Pi and water have been used to identify steps in the E2 half of the reaction cycle affected by the mutations. The study supports the prediction that K501 in the KGAP sequence interacts with adenosine 5'-triphosphate. However, quantitative comparisons of the effect of mutation K501E on the activity with the effects of mutations to an enzyme of known structure that also catalyzes phosphoryl group transfer make a direct role for the positive charge on the side chain of K501 in catalysis by stabilizing the transition state unlikely. No evidence for the predicted interaction between D586 and the hydroxyl groups of ribose was found. However, the data do indicate that the spatial organization of the loop containing the DPPR sequence is critical for phosphorylation of the enzyme. A role for D586 in coordinating the Mg2+ that is required for activity is proposed.


Assuntos
ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Cães , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Técnicas In Vitro , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Ouabaína/farmacologia , Isótopos de Oxigênio , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Ovinos , ATPase Trocadora de Sódio-Potássio/metabolismo
2.
J Biol Chem ; 269(9): 6550-7, 1994 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-8120007

RESUMO

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Primers do DNA , Estabilidade Enzimática , Cinética , Substâncias Macromoleculares , Microssomos/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ouabaína/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae , Ovinos , ATPase Trocadora de Sódio-Potássio/biossíntese
3.
Proc Natl Acad Sci U S A ; 89(7): 2834-8, 1992 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-1313569

RESUMO

Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function.


Assuntos
Ouabaína/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Animais , Western Blotting , Substâncias Macromoleculares , Membranas/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Ovinos , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Estômago/enzimologia
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