title: A 75-kDa Na+,K+-ATPase competitive inhibitor protein isolated from rat brain cytosol binds to a site different from the ouabain-binding site.
abstract: A Na+,K+-ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate precipitation, DEAE anion-exchange chromatography and hydroxyapatite adsorption column chromatography. The purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The inhibitor binds Na+,K+-ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of approximately 700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+-ATPase is hindered in the presence of the inhibitor. It does not affect p-nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD analysis suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor.
authors: Department of Chemistry, Bose Institute, Calcutta, India.
authors: K
authors: K
authors: Roy
authors: A K
authors: AK
authors: Mandal
authors: P C
authors: PC
authors: Sen
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D010042:Ouabain; D014364:Tryptophan; D000254:Sodium-Potassium-Exchanging ATPase