The Na + ,K -ATPase maintains electrochemical gradients for and K that are critical animal cells. Cardiotonic steroids (CTSs), widely used in the clinic recently assigned a role as endogenous regulators of intracellular processes, highly specific inhibitors -ATPase. Here we describe crystal structure phosphorylated pig kidney complex with CTS representative ouabain, extending to 3.4 Å resolution. provides key details on binding, revealing an extensive hydrogen bonding network formed by...

Significance Na + ,K -ATPase is the ion pump responsible for maintenance of electrochemical gradients and K across membrane animal cells. Cardiotonic steroids constitute a broad class specific inhibitors, including drugs clinical importance with multiple physiological effects. The existence several endogenous cardiotonic suggests their involvement in health disease mediated by various signaling pathways, but structure–activity relationships are not yet understood. Using X-ray crystallography...

The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across plasma membrane--a prerequisite for electrical excitability secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present crystal structure a Na(+)-bound K(+)-ATPase as determined at 4.3 Å resolution. Compared with form, large conformational changes are observed in α subunit whereas β γ structures maintained. locations...

Na,K-ATPase is an ubiquitous enzyme actively transporting Na-ions out of the cell in exchange for K-ions, thereby maintaining their concentration gradients across membrane. Since its discovery more than six decades ago Na-pump has been studied extensively and vital physiological role essentially every established. This article aims at providing overview well-established biochemical properties with a focus on isoforms, transport mechanism principle conformations, inhibitors, insights gained...

Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins (Cbls, Kd ≈ 5 fM) but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in following order: IF > TC HC. We investigated interactions of proteins a number ligands: Cbl, fluorescent conjugate CBC, two base-off [pseudo-coenzyme B12 (pB) adenosyl A (fA)], baseless corrinoid cobinamide. Protein−ligand encounter...

Phosphorylation of the alpha-1 subunit rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease activity enzyme in vitro. We have now undertaken an investigation mechanism which this inhibition occurs. Analysis phosphorylation recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that phosphorylated within extreme NH2-terminal domain, on serine-23....

Changes in the absorbance spectrum of aquo-cobalamin (Cbl x OH(2)) revealed that its binding to transcobalamin (TC) is followed by slow conformational reorganization protein-ligand complex (Fedosov, S. N., Fedosova, N. U., Nexø, E., and Petersen, T. E. (2000) J. Biol. Chem. 275, 11791-11798). Two phases were also observed for TC when interacting with a Cbl-analogue cobinamide (Cbi), but not other cobalamins. The phase had no relation ligand recognition, since both Cbl Cbi bound rapidly one...

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with locus tag lmo0841. The purified and detergent-solubilized protein, which we named 1 (LMCA1), performs Ca(2+)-dependent ATP hydrolysis actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite high sequence similarity to sarcoplasmic reticulum (SERCA1a) plasma membrane (PMCA), LMCA1 exhibits important biochemical differences such as low affinity...

Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for uptake Cbl in an organism. Pronounced increase fluorescence upon CBC attachment facilitated detailed kinetic analysis binding. We found that TC had same affinity ( K d = 5 × 10 −15 m ), whereas interaction with highly specific protein IF more complex. For instance, behaved normally partial...

New cardenolides with potent cytotoxic and Na⁺,K⁺-ATPase inhibitory activities were isolated from the ornamental milkweed <italic>Asclepias curassavica</italic>.

Abstract Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition cardiac Na + , K -ATPase (NKA). Bufadienolides constitute one two major classes CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity NKA by native 3β-hydroxy bufalin over 3α-isomer, yet replacing group larger polar groups in same configuration enhances inhibitory potency. Binding 13 C-labelled glycosyl...

Abstract In the healthcare sector, phytocompounds are known to be beneficial by contributing or alleviating a variety of diseases. Studies have demonstrated progressive effects on immune-related diseases and exhibit anticancer effects. Graviola tree is an evergreen with its extracts (leafs seeds) been reported having properties, but precise target action not clear. Using in silico approach, we predicted that annonacin, Acetogenin , active agent found leaf extract (GLE) potentially act as...

Na + ,K -ATPase is responsible for the transport of and K across plasma membrane in animal cells, thereby sustaining vital electrochemical gradients that energize channels secondary transporters. The crystal structure has previously been elucidated using enzyme from native sources such as porcine kidney shark rectal gland. Here, isolation, crystallization first determination bovine a high-affinity E2–BeF 3 − –ouabain complex with bound magnesium are described. Crystals belonging to...

Intrinsic factor (IF50) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF30 and 20 C-terminal glycopeptide IF20. Experiments on binding Cbl to IF30, IF20, IF50 revealed comparable association rate constants (k+Cbl = 4 × 106, 14 26 106 M-1 s-1, respectively), but equilibrium dissociation were essentially different (KCbl 200 μM, 0.2 ≤1 pM, respectively). The smaller fragment, had unexpectedly high affinity for Cbl;...

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence Mg2+ and Na+ or Pi absence alkali cations were investigated by biochemical methods spectrofluorometry employing styryl dye RH421. We characterized phosphoenzyme species their reaction to N-methyl hydroxylamine, which attacks specifically protein−phosphate bond. studied reactions phospho- dephospho-enzymes with vanadate, is a transition-state analogue phosphate this enzyme. On basis substantial differences Pi,...

Binding of aquo-, cyano-, or azidocobalamin (Cbl.OH(2), Cbl.CN, and Cbl.N(3), respectively) to the recombinant human transcobalamin (TC) haptocorrin from plasma was investigated via stopped-flow spectroscopy. Association cobalamins with always proceeded in one step. TC, however, displayed a certain selectivity for ligands: Cbl.CN Cbl.N(3) bound step k(+1) = 1 x 10(8) M(-1) s(-1) (20 degrees C), whereas binding Cbl.OH(2) under same conditions occurred two steps 3 10( 7) (E(a) 30 kJ/mol) k(+2)...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFluorescent styryl dyes as probes for Na,K-ATPase reaction mechanism: significance of the charge hydrophilic moiety RH dyesNatalya U. Fedosova, Flemming Cornelius, and Irena KlodosCite this: Biochemistry 1995, 34, 51, 16806–16814Publication Date (Print):December 1, 1995Publication History Published online1 May 2002Published inissue 1 December 1995https://pubs.acs.org/doi/10.1021/bi00051a031https://doi.org/10.1021/bi00051a031research-articleACS...

Human intrinsic factor (IF) was purified from the recombinant plant Arabidopsis thaliana by affinity chromatography. Cobalamin (Cbl) saturated protein separated gel filtration into peaks I and II, which contained according to SDS electrophoresis 50 kDa full-length IF50 a mixture of two fragments, respectively. Two components peak II were identified as 30 N-terminal peptide IF30 20 C-terminal glycopeptide IF20. Measurements Mw under nondenaturing conditions conducted static light scattering....

Cobalamin (Cbl) is a complex cofactor produced only by bacteria but used all animals and humans. Cyanocobalamin (vitamin B(12), CNCbl) one commonly isolated form of cobalamin. B(12) belongs to large group corrinoids, which are characterized distinct red color conferred the system conjugated double bonds corrin ring retaining Co(III) ion. A unique blue Cbl derivative was hydrolysis CNCbl in weakly alkaline aqueous solution bicarbonate. This corrinoid purified as dark crystals. Its...

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by cis–trans–cis ring-fused steroid core linked to lactone. They have been used in the treatment different medical problems including heart failure, where their inotropic effect on muscle is attributed potent Na+,K+-ATPase inhibition. Their application as drugs, however, has declined recent past years due small safety margin. Since human represented four isoforms expressed tissue-specific manner, one...