Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Lowndes, Joseph M.; Hokin-Neaverson, Mabel; Ruoho, Arnold E.

doi:10.1016/0005-2736(87)90098-8

Cited by 4 publications

(1 citation statement)

References 17 publications

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“…Affinity labeling of the cardiac steroid-binding site has been possible so far only by photoaffinity labeling [4][5][6][7][8]10, 111; attempts to modify amino acids in this site under less harsh labeling conditions with the protein-reactive 2-bromoacetyl ester of strophanthidin [9], digoxigenin, digitoxigenin or gitoxigenin [31] led to inconsistent conclusions. At least the C3 bromoacetyl esters of digoxigenin, digitoxigenin and gitoxigenin were not affinity labels.…”

Section: Discussionmentioning

confidence: 99%

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Antolović

Linder

Hahnen

et al. 1995

European Journal of Biochemistry

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Na+/K+-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidy1 derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylcarbonyl-6-aminocaproate (HDMA), 3-amino-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-0-succinyl-[2-(N-maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37"C, half-maximal inhibition of Na+/K+-ATPase was seen by HDMA at 0.47 pM, by ADHS at 5.8 pM, by IAD at 8 pM and by DSME at 94 pM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+-ATPase. Affinity labeling of the a subunit by 'front door' or 'back door' phosphorylation was only seen with HDMA or ADHS in the range 0.1 pM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain.Tryptic hydrolysis of the HDMA-modified Na+/K+-ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidy1 esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with CyslO4 in the H, transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-'4C]ICH,COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J. Keywords. Na'lK' -ATPase ; N-hydroxysuccinimidyl digoxigenin-3-0-methylcarbonyl-e-aminocaproate ; cardiac glycoside receptor. Na+/K'-ATPase is specifically inhibited by cardioactive steroids [l]. They bind to a specific conformation of Na+/K+-ATPase formed in the catalytic cycle during the hydrolysis of ATP [2, 31. The relatively stable complex with cardiac glycosides is formed via the forward reaction of the enzyme in the presence of Na', Mg" and ATP ('front door' phosphorylation) which forms the cardiac glycoside receptor complex I, or via phosphorylation in the presence of Mgz+ and Pi ('back door' phosphorylation) forming the cardiac glycoside receptor site 11. Enzyme. Na+/K+-ATPase, Na+/K+-transporting ATPase (EC 3.6.1.37).tein-reactive cardiac glycosides have been synthesized with the aim of localizing the cardiac glycoside-binding site [7-111. These studies demonstrated that the 40-kDa N-terminal tryptic fragment of the catalytic a su...

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Section: Discussionmentioning

confidence: 99%

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Antolović

Linder

Hahnen

et al. 1995

European Journal of Biochemistry

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Synthesis of a ¹²⁵I labelled azido‐substituted glibenclamide analogue for photoaffinity labelling of the sulfonylurea receptor

Chudziak

Schwanstecher

Laatsch

et al. 1994

Labelled Comp Radiopharmac

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Action of Drugs on the Erythrocyte Membrane

Deüticke¹,

Grebe²,

Haest³

1990

Blood Cell Biochemistry

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Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Cited by 4 publications

References 17 publications

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Synthesis of a ¹²⁵I labelled azido‐substituted glibenclamide analogue for photoaffinity labelling of the sulfonylurea receptor

Action of Drugs on the Erythrocyte Membrane

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Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Cited by 4 publications

References 17 publications

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na+/K+‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na+/K+‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Synthesis of a 125I labelled azido‐substituted glibenclamide analogue for photoaffinity labelling of the sulfonylurea receptor

Action of Drugs on the Erythrocyte Membrane

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Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na⁺/K⁺‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin

Synthesis of a ¹²⁵I labelled azido‐substituted glibenclamide analogue for photoaffinity labelling of the sulfonylurea receptor