Ouabain, binding

The reactivity of the sodium pump isoforms toward ouabain can significantly differ between certain isoenzymes. The al-isoform from rat is reported to be 100-fold more resistant to ouabain than a2- or a3-isoform. In other species like rabbit, pig, dog or human these differences are not as distinct as in rat, due to a more sensitive al-isoform. At least two different types of ouabain binding sites are reported for the Na+/fC+-ATPase [1]. 

Desaiah D, Gilliland IK, Ho IK, et al. 1980a. Inhibition of mouse brain synaptosomal ATPases and ouabain binding by chlordecone. Toxicol Lett 6 275-285. 

A classical example of sequestration is the monarch butterfly Danaus plexip-pus (Danainae) which feeds on leaves oiAsclepias curassavica (Asclepiadaceae) and sequesters cardenolides such as calotropin (158) (Fig. 29). This molecule was shown to afford the butterfly an efficient protection against birds [150-152]. It was also demonstrated that its resistance to cardiac glycosides is due to a single amino acid mutation in the ouabain-binding site of the Na+/K+ ATPase [153,154]. 

Other Proteins The ouabain-binding site on (Na /K -adenosine-5 -triphosphatase, 46, 523 penicillin isocyanates for /3-lactamase, 46, 531 active site-directed addition of a small group to an enzyme the ethylation of ludferin, 46, 537 mandelate racemase, 46, 541 d imethylpyrazole carboxamidine and related derivatives, 46, 548 labeling of catechol O-methyltransferase with N-haloace-tyl derivatives, 46, 554 affinity labeling of binding sites in proteins by sensitized photooxidation, 46, 561 bromocolchicine as a iabei for tubuiin, 46, 567. 

A full consideration of the mechanism of the sodium pump requires an account of the role of the lipid, the binding sites for Na+, K+, Mg2+ and ATP, the mechanism of hydrolysis of ATP and the way in which this is coupled to the transport of the cation. In addition it should be noted that the enzyme also functions as a K+-dependent phosphatase, a reaction usually studied with p-nitrophenyl phosphate as substrate. Studies with inhibitors have been informative, notably with ouabain and with vanadate. Ouabain binds at one site per pump and so has been of value in quantitatively defining the enzyme in various preparations. 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

The Na-K ATPase, in the plasma membrane of mammalian cells, is inhibited when ouabain binds to the a subunit that is phosphorylated during catalysis.21 372 Unlike HGPRT" and TK"/", the ouabain-resistant phenotype (assigned to chromosome 3 in the mouse)218 is dominant, stable, and characterized by a reduced uptake of 86Rb and 1 2K in the presence of ouabain and by reduced binding of ouabain to the plasma membrane.21 Ouabain resistance can be induced... 

Two inhibitors of the exchange mechanism and ATP hydrolysis are ouabain and vanadate. Ouabain binds only to that portion of ATPase that would be exposed to the extracellular surface in situ. It appears to stimulate the incorporation of orthophosphate into the same site that is phosphorylated by ATP under normal conditions, but ouabain inhibits all pump and hydrolytic... 

The a-subunit carries the ATP binding site [88], the phosphorylation site [89-91] and the specific sites for inhibition by )V-ethylmaleimide [92] and ouabain [93-95], Since ouabain and ATP bind at opposite sides of the membrane, this implies that the a-subunit must traverse the membrane. Antibodies have been prepared against the a- and /8-subunits [96]. Those against the a-subunit inhibited phosphorylation but not ouabain binding, whereas those against the )S-subunit inhibited ouabain binding but not the phosphorylation. This means that, since ouabain binds to the outer membrane side, the /8-subunits must, at least partially, be oriented to that side. 

In terms of the often used pH]-ouabain radioligand potency assay [91,92], this clearly means that an inhibitory ligand effect on pH]-ouabain binding to the enzyme does not necessarily indicate its competition with ouabain for the digitalis-binding matrix as has often been presumed. In conclusion, all data extracted from the radioligand potency assay, as for example, for prednisolone bisguanylhydrazone [93,94] or chlormadinone acetate [91], should be evaluated with this reservation in mind. 

Energy transfer experiments with anthroylouabain indicated that a tryptophan residue is located close to the ouabain binding site [120]. 

Inhibition of ouabain binding to membrane Na, K -ATPase and cross-reactivity with digoxin antibody in a radioimmunoassay from a pleurisy root aqueous extract were observed in vitro (Longerich et al. 1993). 

O Brien WJ, Lingrel JB, Wallick ET (1994) Ouabain binding kinetics of the rat alpha two and alpha 3 isoforms of the sodium-potassium adenosine triphosphate. Arch Biochem Biophys 310 32-39... 

Strophanthidin 3,5-bis-4-benzoylbenzoate Ouabain binding site of brain Na, K+-ATPase Loss of enzyme activity on photolysis but much nonspecific attachment 131... 

Conditions for Photoaffinity Labeling of the Ouabain-Binding Site of... 

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