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Epitope and mimotope for an antibody to the Na, K-ATPase.

ABSTRACT: The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.

SUBMITTER: Pacholczyk T

PROVIDER: S-EPMC2143741 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Epitope and mimotope for an antibody to the Na, K-ATPase.

Pacholczyk T T Sweadner K J KJ

Protein science : a publication of the Protein Society 19970701 7

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on ...[more]

PMID: 9232655

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Project description:The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na(+)-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na(+)-Na(+) exchange mode were measured at 23 degrees C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na(+) concentrations ([Na](o)) between 36 and 145 mM at membrane potentials (V(M)) from -160 to +80 mV. Analysis of charge-V(M) curves showed that the midpoint potential of charge distribution was shifted toward more positive V(M) both by increasing [ADP] at constant Na(+)(o) and by increasing [Na](o) at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na](o). The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V(M) but decreased it at depolarizing V(M) as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na(+)-Na(+) exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K(d) of approximately 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s(-1) for k(2), a rate constant that groups Na(+) deocclusion/release and the enzyme conformational transition E(1) approximately P --> E(2)-P; a value of 162 s(-1)M(-1) for k(-2), a lumped second-order V(M)-independent rate constant describing the reverse reactions; and a Hill coefficient of approximately 1 for Na(+)(o) binding to E(2)-P. The results are consistent with electroneutral release of ADP before Na(+) is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.

Project description:BackgroundWhen cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, the ATPase involved in Na+ transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na+ transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions.ResultsThe purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F1F0-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na+ in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP, respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP) but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes.ConclusionThe data demonstrate that Na+-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na+ pump which transports only Na+ upon hydrolysis of ATP. A secondary event, Na+- and ATP-dependent H+ efflux from proteoliposomes, is driven by the electric potential generated by Na+-stimulated ATPase.

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Epitope and mimotope for an antibody to the Na, K-ATPase.

Publications

Epitope and mimotope for an antibody to the Na, K-ATPase.

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