Purification, characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol.
A protein isolated from goat testis cytosol is found to inhibit Na+,K+-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I50) occurs is in the nanomolar range. The inhibitor seems to bind Na+,K+-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na+,K+-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.
Mandal
A K
AK
Department of Chemistry, Bose Institute, Calcutta, India.
Roy
K
K
Sil
P C
PC
Yadav
S P
SP
Sen
P C
PC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006041:Goats; D008297:Male; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013737:Testis / Q000737:chemistry* / Q000648:ultrastructure
D000596:Amino Acids; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011506:Proteins; C008644:nitrophenylphosphate; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase