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.