Enzyme inhibitors ouabain

That the sodium pump operates in cestodes has been clearly shown in H. diminuta and the same inhibitors (ouabain and phlorizin) which inhibit the system in the mucosal cells are also effective in cestodes (Figs 2.2 and 2.4). One major difficulty, however, in applying the intestinal cell model to the cestode tegument is that the enzyme Na+/K+-ATPase, which is involved in the former, has only been identified with certainty in E. granulosus (491) and not in any other species (878). 

We have found that the ecto-ATPase is optimally stimulated by Mn at concentrations of 3 x 10" or below, but we have also observed some exceptions (for instance, in mouse neuroblastoma where 10 M Ca was most effective and Mn did not stimulate). Mn concentrations in excess of 3 x 10 M are inhibitory. The ecto-ATPase seems widely distributed in eukaryotic cells. There has been an earlier report that the enzyme was not present on the surface of intact neurons (CUMMINS HYDEN, 1962) but it certainly is present on the surface of intact neuroblastoma cells (TRAMS 6e LAUTER, 1974 STEFANOVIC et ad, 1976b), Conventional sulfhydryl compounds or inhibitors have little effect on the enzyme and ouabain is not inhibitory. We have found that ecto-ATPase of cultured CNS cells was inhibited by certain phenothiazine derivatives. Micromolar concentrations of thiazines and tricyclic antidepressants were inhibitory in rat leukocytes (MEDZIHRADSKY et, 1975). There has been one report that adipocyte membrane Mg ATPase was markedly stimulated by insulin and by Concanavalin A (JARRETT Sc SMITH, 1974). ... 

The steroid derivative ouabain (pronounced wah -bane from waa bayyo, Somali for arrow poison ) is a potent and specific inhibitor of the Na+K+ ATPase. Oubain binds preferentially to the form of the enzyme that is open to the extracellular side, locking in two Na+ ions and preventing the changes of conformation necessary to ion transport. Another very potent toxin, palytoxin (produced by a coral on the Hawaiian shoreline), also targets the Na+K+ ATPase, but it binds... 

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... 

Fig. 6. A, ChE activity of homogenates from the cerebrum (white columns) and retina (hatched columns) of atropinized male rats 16 h after subcutaneous administration of 2-MPAM-ES iodide (0.325 mg/kg). Two hours prior to the administration of the inhibitor the animals were pretreated with subcutaneous injections of 2,4-dinitrophenol (10 mg/kg), ouabain (1 mg/kg), and X-ray irradiation, 5,000 rads, through the head. Enzyme activities are expressed as percentages of the normal. Erythrocyte ChE activity (not shown on the figure) was reduced to 24% (see also Fig. 3). B, Columns denoting ChE in the cerebrum and retina as in A, but from animals pretreated with paraoxon (0.3 mg/kg) intravenously. The left pair of columns represents control animals which were given no subsequent reactivator therapy, whereas the following 3 pairs of columns show the effect of the enzyme reactivators indicated on figure. The ChE reactivation is seen to be more pronounced in the retina than in the cerebrum. Reactivation of erythrocyte ChE (not shown) was materially complete in these experiments. For further details (reactivator dosage, number and time-spacing of doses, etc.) see text.

Other inhibitors of the enzyme also had similar effects on the ac-stimu-lated activity. Ouabain (50% inhibition at 0.35 xM), oligomycin (8.0 xM), ouabagenin (3 jlM), and vanadate (20 jlM) all inhibited the ac-stimulated pumping activity. 4,4 -Bis(isothiocyano)-2,2 -distilbenesulfonate (DIDS), an inhibitor of band 3 protein-mediated Li+/Na+ exchange, had no effect, nor did phloretin at micromolar concentrations. These results show that Na, K-ATPase is responsible for the observed effects. 

Na /K -ATPase, a key enzyme in the regulation of intracellular levels of these cations in most organisms, has been demonstrated in the tegument of E. granulosus, but has not yet been detected in other cestodes (3). The effect of ouabain, a specific inhibitor of this enzyme, on amino acid absorption by H. diminuta suggests that Na /K -ATPase is present and functional in other speeies. 

A first answer to the question of the nature of the cation pump has been supplied by Skou [3]. He reasoned that making the energy of ATP available to cation transport would require an ATPase, an enzyme which splits ATP into A DP and inorganic phosphate. Since such an ATPase would have to make this energy available to the translocation of Na+ and K ions, he assumed that it would require these cations for activity. In a study of a crab nerve particulate fraction he was able to demonstrate a Mg-activated ATPase activity, which was increased considerably upon simultaneous addition of Na and to the assay medium. In addition he showed that the digitalis glycoside ouabain, which had some years earlier been found to be a powerful and specific inhibitor of active eation transport in erythrocytes [4], completely inhibited the additional ATPase activity [5]. From these two observations Skou concluded that this ouabain-sensitive, (Na + K )-stimulated ATPase activity (henceforth called Na-K ATPase) might be identical with or be part of the cation transport system. 

The enzyme can also be inhibited by some other agents, but the mechanism of their action has not been elucidated. These agents are Zn [54,62], fluoride [54,62], vanadate [83] and dipicrylamine [66]. Other agents such as thiocyanate and ouabain, which are inhibitors of the anion-sensitive ATPase and (Na +K )-ATPase, respectively, have no effect on the (K + H )-ATPase [54], Two out of five antibody preparations against purified (K + H )-ATPase inhibit the ATPase activity for 80% and the K +-phosphatase activity for 35%, while the three other preparations have no effect on the enzyme activity [70], suggesting a heterogeneity of these preparations. 

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