Identification, purification and partial characterization of a 70kDa inhibitor protein of Na(+)/K(+)-ATPase from cytosol of pulmonary artery smooth muscle.
OBJECTIVE
We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle.


METHODS
(i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor.


RESULTS
We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer.


CONCLUSIONS
The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Kuntal
K
Dey
Tapati
T
Chakraborti
Soumitra
S
Roy
Biswarup
B
Ghosh
Pulak
P
Kar
Sajal
S
Chakraborti
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010042:Ouabain / Q000302:isolation & purification* / Q000378:metabolism*; D020033:Protein Isoforms; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D020033:Protein Isoforms; C083016:sodium-potassium ATPase inhibitory factor; D010042:Ouabain; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase