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Purification, characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

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Abstract

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.

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References

  1. Lutsenko S, Kaplan JH: In: A. Scarpa, E. Carafoli, S. Papa (eds). Ionmotive ATPases: Structure, Function, and Regulation. Ann NY Acad Sci 671: 147–155, 1992

  2. Schurmans-Stekhoven F, Bonting SL: Transport adenosine triphosphatases: Properties and functions. Physiol Rev 61: 1–76, 1981

    Google Scholar 

  3. Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T: Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 44: 377–399, 1992

    Google Scholar 

  4. Chandra S, Adhikary G, Sikdar R, Sen PC: An Na+/K+-ATPase inhibitor protein from rat brain cytosol. Biochim Biophys Acta 1144: 33–38, 1993

    Google Scholar 

  5. Shirley C, Vesely DL: Kaliuretic peptide: The most potent inhibitor of Na+,K+-ATPase of the atrial natriuretic peptides. Endocrinology 136: 2033–2039, 1995

    Google Scholar 

  6. Chih-Fang C, Shen-Tai C, Hellen J, Wen-Chang C: The porcine sperm motility inhibitor is identical to β-microseminoprotein and is a competitive inhibitor of Na+,K+-ATPase. Biochem Biophys Res Commun 218: 623–628, 1996

    Google Scholar 

  7. Bhattacharyya D, Sen PC: Purification and functional characterization of a low molecular-mass Na+,K+-ATPase inhibitor protein from rat brain cytosol. Eur J Biochem 244: 829–834, 1997

    Google Scholar 

  8. Roy K, Mandal AK, Sen PC: A 75-kDa Na+,K+-ATPase competitive inhibitor protein isolated from rat brain cytosol binds to a site different from the ouabain-binding site. Eur J Biochem 261: 84–89, 1999

    Google Scholar 

  9. Robinson JD: Kinetic studies on a brain microsomal adenosine triphosphatase: Evidence suggesting conformational changes. Biochemistry 6: 3250–3258, 1967

    Google Scholar 

  10. Sen PC, Kapakos JG, Steinberg M: Modification of (Na++ K+)-dependent ATPase by fluorescein isothiocyanate: Evidence for the involvement of different amino groups at different pH values. Arch Biochem Biophys 211: 652–662, 1981

    Google Scholar 

  11. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976

    Google Scholar 

  12. Lammeli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970

    Google Scholar 

  13. Morrissey JH: Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal Biochem 117: 307–310, 1981

    Google Scholar 

  14. Van Winkle WB, Allen JG, Schwartz A: The nature of the transport ATPase-digitalis complex: III. Rapid binding studies and effects of ligands on the formation and stability of magnesium plus phosphateinduced glycoside-enzyme complex. Arch Biochem Biophys 151: 85–92, 1972

    Google Scholar 

  15. Post RL, Sen AK: 32P-labeling of a (Na++ K+)-ATPase intermediate. Meth Enzymol 10: 773–776, 1967

    Google Scholar 

  16. De Lores Arnaiz GR, De Lima MADG, Girardi E: Different properties of two brain extracts separated sephadex G-50 that modify synaptosoma activities. Neurochem Res 13: 229–235, 1988

    Google Scholar 

  17. Kairane C, Zilmer M, Mutt V, Sillard R: Activation of Na,K-ATPase by an endogenous peptide, PEC-60. FEBS Lett 345: 1–4, 1994

    Google Scholar 

  18. Bhattacharyya D, Sen PC: Purification and functional characterization of a low-molecular-mass Ca2+, Mg2+-and Ca2+-ATPase modulator protein from rat brain cytosol. Biochem J 330: 95–101, 1998

    Google Scholar 

  19. Phillis JW: In: S. Fedoroff, L. Hertz (eds). Cell, Tissue and Organ Cultures in Neurobiology. Academic Press, New York, 1978, pp 93–97

    Google Scholar 

  20. Cantley LC Jr, Cantly LG, Josephson L: A characterization of vanadate interactions with the (Na,K) ATPase; mechanistic and regulatory implications. J Biol Chem 253: 7361–7368, 1978

    Google Scholar 

  21. Abbott AJ, Holoubek CG, Martin RA: Inhibition of Na+,K+-ATPase by the cardenolide 6′—O-(E-4-hydroxycinnamoyl) desglucouzarin. Biochem Biophys Res Commun 251: 256–259, 1998

    Google Scholar 

  22. Karlish SJD: In: Na+,K+-ATPase: Structure and Kinetics. Academic Press, New York, 1979, pp 115–128

    Google Scholar 

  23. Sen PC, Meng Ai Z, Ray TK: Bi-layer orientation of membrane bound-NH2 groups across microsomal vesicle and their role in the function of gastric potassium stimulated ATPase. Arch Biochem Biophys 205: 340–351, 1980

    Google Scholar 

  24. Robinson JD, Flashner MS: The (Na++ K+)-activated ATPase: Enzymatic and transport properties. Biochim Biophys Acta 549: 145–176, 1979

    Google Scholar 

  25. Uesugi S, Yamazoe S: Presence of sodium-potassium ion stimulated ATPase in boar spermatozoa. Nature 209: 403, 1966

    Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Calcutta -, 700 009, India

    Atin K. Mandal, Koushik Roy & Parimal C. Sen

  2. The Cleveland Clinic Foundation, Ohio, USA

    Parames C. Sil & Satya P. Yadav

Authors
  1. Atin K. Mandal
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  2. Koushik Roy
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  3. Parames C. Sil
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  4. Satya P. Yadav
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  5. Parimal C. Sen
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Correspondence to Parimal C. Sen.

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Mandal, A.K., Roy, K., Sil, P.C. et al. Purification, characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol. Mol Cell Biochem 223, 7–14 (2001). https://doi.org/10.1023/A:1017527026796

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