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Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

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Abstract

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (≈81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.

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References

  1. Lowenstein JM: Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol Rev 52: 382–414 1972

    Google Scholar 

  2. Fishbein WN, Sabina RL, Ogasawara N, Holmes EW: Immunologic evidence for three forms of AMP deaminase (AMPD) in mature skeletal muscle. Biochim Biophys Acta 1163: 97–104, 1993

    Google Scholar 

  3. Mahnke-Zizelman DK, Sabina RL: Cloning of human AMP deaminase isoform E cDNAs. Evidence for a third AMPD gene exhibiting alternatively spliced 5′-exons. J Biol Chem 267: 20866–20877 1992

    Google Scholar 

  4. Morisaki T, Holmes EW: Functionaily distinct elements are required for expression of the AMPD1 gene in myocytes. Mol Cell Biochem 13: 5854–5860, 1993

    Google Scholar 

  5. Ogasawara N, Goto H, Watanabe T: Isozymes of rat AMP deaminase. Biochim Biophys Acta 403: 530–537, 1975

    Google Scholar 

  6. Jenkins RL, McDaniel HG, Atkins L: Changes in AMP deaminase activities in the hearts of diabetic rats. Biochim Biophys Acta 1077: 379–384, 1991

    Google Scholar 

  7. Thakkar JK, Janero DR, Yarwood C, Sharif HM, Hreniuk D: Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium. Biochem J 290: 335–341, 1993

    Google Scholar 

  8. Mineo I, Clarke PRH, Sabina RL, Holmes EW: A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5′ splice donor site not present in the primary transcript of AMPDI. Mol Cell Biol 10: 5271–5278, 1990

    Google Scholar 

  9. Thakkar JK, Janero DR, Yarwood C, Sharif HM: Modulation of mammalian cardiac AMP deaminase by protein kinase C-mediated phosphorylation. Biochem J 291: 523–527, 1993

    Google Scholar 

  10. Thakkar JK, Janero DR, Sharif HM, Hreniuk D, Yarwood C: Cardiac adenylate deaminase: molecular, kinetic, and regulatory properties under phosphate-free conditions. Biochem J 300: 359–363, 1994

    Google Scholar 

  11. Thompson JL, Sabina RL, Ogasawara N, Riley DA: AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle. J Histochem Cytochem 40: 931–946, 1992

    Google Scholar 

  12. Ogasawara N, Goto H, Yamada Y, Watanabe T, Asano T: AMP deaminase isozymes in human tissues. Biochim Biophys Acta 714: 298–306, 1982

    Google Scholar 

  13. Ogasawara N, Goto H, Yamada Y: AMP deaminase isozyme in rabbit red and white muscles and heart. Comp Biochem Physiol 76B: 471–473, 1983

    Google Scholar 

  14. Janero DR, Yarwood C: Oxidative modulation and inactivation of rabbit cardiac adenylate deaminase. Biochem J 306: 421–427, 1995

    Google Scholar 

  15. Lindberg B, Klenow H, Hansen K: Some properties of partiaily purified mammalian adenosine kinase. J Biol Chem 242: 350–356, 1967

    Google Scholar 

  16. Sandhu G, Burrier A, Janero DR: Adenosine deaminase inhibitors attenuate ischemic injury and preserve energy balance in isolated guinea pig heart. Am J Physiol 265: H1249-H1256, 1993

    Google Scholar 

  17. Janero DR, Hreniuk D, Sharif HM: Hydroperoxide-induced oxidative stress impairs heart muscle-cell carbohydrate metabolism. Am J Physiol 266: C179-C188, 1994

    Google Scholar 

  18. Bradford M: A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of proteindye binding. Anal Biochem 72: 248–254, 1976

    Google Scholar 

  19. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 5340–5354, 1979

    Google Scholar 

  20. Bjorck L, Kronvall G: Purification and some properties of Streptococcal protein G, a novel IgG-binding reagent. J Immunol 133: 969–974, 1984

    Google Scholar 

  21. Merkler DJ, Schramm VL: Catalytic mechanism of yeast adenosine 5′ monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects. Biochemistry 32: 5792–5799, 1993

    Google Scholar 

  22. Askari A, Franklin JE: Effects of monovalent cations and ATP on erythrocyte AMP deaminase. Biochim Biophys Acta 110: 162–173, 1965

    Google Scholar 

  23. Nathans GR, Chang D, Deuel TF: AMP deaminase from human erythrocytes. Methods Enzymol 51: 497–502, 1978

    Google Scholar 

  24. Ogasawara N, Goto H, Yamada Y, Watanabe T: Distribution of AMP deaminase isozymes in rat tissues. Eur J Biochem 87: 297–304, 1978

    Google Scholar 

  25. Van Kuppevelt TH, Veerkamp JH, Fishbein WN, Ogasawara N, Sabina RL: Immunolocalization of AMP-deaminase isozymes in human skeletal muscle and cultured muscle cells: concentration of isoform M at the neuromuscular junction. J Histochem Cytochem 42: 861–868, 1994

    Google Scholar 

  26. Bausch-Jurken MT, Mahnke-Zizelman DK, Morisaki T, Sabina RL: Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA. J Biol Chem 267: 22407–22413, 1992

    Google Scholar 

  27. Ronca F, Ranieri-Raggi M, Brown PE, Moir AJG, Raggi A: Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase. Biochim Biophys Acta 1209: 123–129, 1994

    Google Scholar 

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Author notes

  1. Craig Yarwood

    Present address: Medical Education and Publications Department, Ciba-Geigy Corporation, 07901, Summit, NJ, USA

Authors and Affiliations

  1. Research Department, Ciba Pharmaceuticals, Summit, New Jersey, USA

    Jay K. Thakkar, David R. Janero, Haamid M. Sharif & Craig Yarwood

Authors
  1. Jay K. Thakkar
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  2. David R. Janero
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  3. Haamid M. Sharif
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  4. Craig Yarwood
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Thakkar, J.K., Janero, D.R., Sharif, H.M. et al. Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody. Mol Cell Biochem 145, 177–183 (1995). https://doi.org/10.1007/BF00935490

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  • DOI: https://doi.org/10.1007/BF00935490

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