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α-Lactalbumin and (Calcium-Binding) Lysozyme

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Calcium-Binding Protein Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 172))

Abstract

Lysozyme and α-lactalbumin have undoubtedly evolved from a common ancestor because of the similarity of their amino acid sequences (1). Studies of α-lactalbumin and lysozyme published before 1990 have been reviewed by McKenzie and White (2). α-Lactalbumin, which is a major milk component of milk whey, is a calcium-binding metalloprotein (3). It is the so-called B component of lactose synthase (4) and acts as a specificity modifier of galactosyltransferase to convert it to lactose synthase (5). The original discovery of its calcium-binding property was made when an effect of ethylenediaminetetracetic acid (EDTA) on the conformational stability of bovine α-lactalbumin was demonstrated. With the addition of 1 mM EDTA, the unfolding temperature decreased by 20-32 degrees. α-Lactalbumin was confirmed by flame spectrophotometry to bind one calcium ion tightly (3). The binding constant for calcium ions to α-lactalbumin has been determined with a variety of methods and the results have been summarized by Kronman (6).

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References

  1. Brew, K., Castellino, F. J., Vanaman, T. C. and Hill, R. L. (1970) The complete amino acid sequence of bovine α-lactalbumin. J. Biol. Chem. 245, 4570–4582.

    CAS  PubMed  Google Scholar 

  2. McKenzie, H. A. and White, F. H., Jr. (1991) Lysozyme and α-lactalbumin: structure, function, and interrelationships. Adv. Protein Chem. 41, 173–315.

    Article  CAS  PubMed  Google Scholar 

  3. Hiraoka, Y., Segawa, T., Kuwajima, K., Sugai, S., and Murai, N. (1980) α-Lactal-bumin: a calcium metalloprotein. Biochem. Biophys. Res. Commun. 95, 1098–1104.

    Article  CAS  PubMed  Google Scholar 

  4. Brodbeck, U., Denton, W. L., Tanahashi, N., and Ebner, K. E. (1967) The isolation and identification of the B protein of lactose synthetase as α-lactalbumin. J. Biol. Chem. 242, 1391–1397.

    CAS  PubMed  Google Scholar 

  5. Brew, K., Vanaman, T. C., and Hill, R. L. (1970) The role of α-lactalbumin and the A protein in lactose synthase: a unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. USA 59, 491–497.

    Article  Google Scholar 

  6. Kronman, M. J. (1989) Metal-ion binding and the molecular conformational properties of α-lactalbumin. Crit. Rev. Biochem. Mol. Biol. 24, 565–667.

    Article  CAS  PubMed  Google Scholar 

  7. Kuwajima, K., Nitta, K., Yoneyama, M., and Sugai, S. (1976) Three-state denatur-ation of α-lactalbumin by guanidine hydrochloride. J. Mol. Biol. 106, 359–373.

    Article  CAS  PubMed  Google Scholar 

  8. Segawa, T. and Sugai, S. (1983) Interactions of divalent metal ions with bovine, human and goat α-lactalbumin. J. Biochem. 93, 1321–1328.

    CAS  PubMed  Google Scholar 

  9. Kuwajima, K., Mitani, M., and Sugai, S. (1989) Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+binding on the folding kinetics of α-lactalbumin. J. Mol. Biol. 206, 547–561.

    Article  CAS  PubMed  Google Scholar 

  10. Stuart, D. I., Acharya, K. R., Walker, N. P. C., Smith, S. G., Lewis, M., and Philips, D. C. (1986) α-Lactalbumin possesses a novel calcium binding loop. Nature 324, 84–87.

    Article  CAS  PubMed  Google Scholar 

  11. Nitta, K. Tsuge, H., Sugai, S., and Shimazaki, K. (1987) The calcium-binding property of equine lysozyme. FEBS Lett. 223, 405–408.

    Article  CAS  PubMed  Google Scholar 

  12. Nitta, K., Tsuge, H., Shimazaki, K., and Sugai S. (1988) Calcium-binding lysozymes. Biol. Chem. 369, 671–675.

    CAS  Google Scholar 

  13. Nitta, K. and Sugai, S. (1989) The evolution of lysozyme and α-lactalbumin. Eur. J. Biochem. 182, 111–118.

    Article  CAS  PubMed  Google Scholar 

  14. Grobler, J. A., Rao, K. R., Pervaiz, S., and Brew, K. (1994) Sequences of two highly divergent canine type c lysozymes: implications of the evolutionary origins of the lysozyme/α-lactalbumin superfamily. Arch. Biochem. Biophys. 313, 360–366.

    Article  CAS  PubMed  Google Scholar 

  15. Prager, E. M. and Jolles, P. (1996) Animal lysozymes c and g: an overview, in Lysozymes: Model Enzymes in Biochemistry and Biology (Jolles, P., ed.), Bazel-Boston-Berlin, Birkhauzer Verlag, pp.9–31.

    Google Scholar 

  16. Guss, J. M., Messer, M., Costello, M., Hardy, K., and Kumar, V. (1997) Structure of the calcium-binding echidna milk lysozyme at 1.9 Å resolution. Acta Crystallogr. 53, 355–363.

    CAS  Google Scholar 

  17. Mizuguchi, M., Nara, M., Kawano, K., and and Nitta, K. (1997) FT-IR study of the Ca2+-binding to bovine α-lactalbumin. Relationships between the types of coordination and characteristics of the bands due to the Asp COO- groups in the Ca2+-binding site. FEBS Lett. 417, 153–156.

    Article  CAS  PubMed  Google Scholar 

  18. Mizuguchi, M., Yue K., Kawano, K., Hiraoki, T., and Nitta, T. (1997) FT-IR Studies on the coordination of the side-chain COO- groups to Ca2+in equine lysozyme. Eur. J. Biochem. 250, 72–76.

    Article  CAS  PubMed  Google Scholar 

  19. Nara, M., Tasumi, M., Tanokura, M., Hiraoki, T., Yazawa, M., and Tsutsumi, A. (1994) Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin (pI = 4. 10). FEBS Lett 349, 84–88.

    Article  CAS  PubMed  Google Scholar 

  20. Godovac-Zimmermann, J., Conti, A., and Napolitano, L. (1988) The primary structure of donkey (equus asinus) lysozyme contains the Ca(II) binding site of α-lactalbumin. Biol. Chem. 369, 1109–1115.

    CAS  Google Scholar 

  21. Teahan, C. G., McKenzie, H. A., Shaw, D. C., and Griffiths, M. (1991) The isolation and amino acid sequences of echidna (tachyglossus aculeatus) milk lysozyme I and II. Biochem. Int. 24 85–95.

    CAS  PubMed  Google Scholar 

  22. Kikuchi, M., Kawano, K., and Nitta, K. (1998) Calcium-binding and structural stability of echidna and canine lysozymes. Protein Sci. 7, 2150–2155.

    Article  CAS  PubMed  Google Scholar 

  23. Nitta, K., Tsuge, H., and Iwamoto, H. (1993) Comparative study of the stability of the folding intermediates of the calcium-binding lysozymes. Int. J. Protein Res. 41, 118–123.

    CAS  Google Scholar 

  24. Mizuguchi, M., Arai, M., Yue Ke, Nitta, K., and Kuwajima K. (1998) Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy. J. Mol. Biol. 283, 265–277.

    Article  CAS  PubMed  Google Scholar 

  25. Van Dael, H., Haezebrouck, P., Morozova, L., Ariko-Muendel, C., and Dobson, C. M. (1993) Partially folded states of equine lysozyme. Structural characterization and significance for protein folding. Biochemistry 32, 11,886–11,894.

    Article  PubMed  Google Scholar 

  26. Griko, Y. V., Freire, E., Privalov, G., Van Dael, H., and Privalov, P. L. (1995) The unfolding thermodynanics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme. J. Mol. Biol. 252, 447–459.

    Article  CAS  PubMed  Google Scholar 

  27. Morozova, L., Haynie, D. T., Ariko-Muendel, C., Van Dael, H., and Dobson, C. M. (1995) Structural basis of the stability of a lysozyme molten globule. Nat. Struct. Biol. 2, 871–875.

    Article  CAS  PubMed  Google Scholar 

  28. Morozova, L., Ariko-Muendel, C., Haynie, D. T., Emelyanenko, V. I., Van Dael, H., and Dobson, C. M. (1997) Structural characterisation and comparison of the native andA-states of equine lysozyme. J. Mol. Biol. 268, 903–921.

    Article  Google Scholar 

  29. Van Dael, H. (1998) Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding. Cell. Mol. Life Sci. 54, 1217–1230.

    Article  PubMed  Google Scholar 

  30. Yao, M., Tanaka, I., Hikichi, K., and Nitta, K. (1992) Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme. J. Biochem. 111, 1–3.

    CAS  PubMed  Google Scholar 

  31. Tsuge, H., Ago, H., Noma, M., Nitta, K., Sugai, S., and Miyano, M. (1992) Crystallographic studies of a calcium binding lysozyme from equine milk at 2.5 Å resolution. J. Biochem. 111, 141–143.

    CAS  PubMed  Google Scholar 

  32. Inaka, K., Kuroki, R., Kikuchi, M., and Matsushima, M. (1991) Crystal structures of the apo-and holomutant human lysozymes with an introduced Ca2+binding site. J. Biol. Chem. 266, 20,666–20,671.

    CAS  PubMed  Google Scholar 

  33. Kuroki, R., Nitta, K., and Yutani, K. (1992) Thermodynamic changes in the binding of Ca2+to a mutant human lysozyme(D86/92).J. Biol. Chem. 267, 24,297–24,301.

    CAS  PubMed  Google Scholar 

  34. Kim, S. and Baum, J. (1998) Electrostatic interactions in the acid denaturation of α-lactalbumin determined by NMR. Protein Sci. 7, 1930–1938.

    Article  CAS  PubMed  Google Scholar 

  35. Nitta, K. and Watanabe, A. (1991) Determination of calcium ions tightly bound to proteins. J. Chromatogr. 585, 173–176.

    Article  CAS  PubMed  Google Scholar 

  36. Tsien, R. Y. (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 2396–2404.

    Article  CAS  PubMed  Google Scholar 

  37. Koshiba, T., Tsumoto, K., Masaki, K., Kawano, K., Nitta, K., and Kumagai, I. (1998) Thermal study of mutant human lysozymes with partially introduced Ca2+binding sites by using efficient refolding system from inclusion bodies. Protein Eng. 11, 683–690.

    Article  CAS  PubMed  Google Scholar 

  38. Koshiba, T., Hayashi, T., Ishido, M., Kumagai, I., Ikura, T., Kawano, K., et al. (1999) Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein. Protein Eng. 12, 429–435.

    Article  CAS  PubMed  Google Scholar 

  39. Tsuge, H., Koseki, K., Miyano, M., Shimazaki, K., Chuman, T., Matsumoto, T., et al. (1991) A structural study of calcium-binding equine lysozyme by twodimensional 1H-NMR. Biochim. Biophys. Acta 1078, 77–84.

    Article  CAS  PubMed  Google Scholar 

  40. Aramini, J. M., Drakenberg, T., Hiraoki, T., Nitta, K., Yue, K., and Vogel, H. J. (1992) Calcium-43 NMR studies of calcium binding lysozymes and α-lactalbumin. Biochemistry 31, 6761–6768.

    Article  CAS  PubMed  Google Scholar 

  41. Acharya, K. R., Stuart, D. I., Walker, N. P. C., Lewis, M., and Philips, D. C. (1989) Refined structure of baboon α-lactalbumin at 1.7 Å resolution comparison with C-type lysozyme. J. Mol. Biol. 208, 99–127.

    Article  CAS  PubMed  Google Scholar 

  42. Acharya, K. R., Ren, J., Stuart, D. I., Philips, D. C., and Fenna, R. E. (1991) Crystal structure of human α-lactalbumin at 1.7 Å resolution. J. Mol. Biol. 221, 571–581.

    Article  CAS  PubMed  Google Scholar 

  43. Pike, A. C. W., Brew, K., and Acharya, K. R. (1996) Crystal structures of guineapig, goat and bovine α-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. Structure 4, 691–703.

    Article  CAS  PubMed  Google Scholar 

  44. Aramini, J. M., Hiraoki, T., Yue, K., Nitta, K., and Vogel, H. J. (1995) Cadmium-113 NMR studies of bovine and human α-lactalbumin and equine lysozyme. J. Biochem. 117, 623–628.

    CAS  PubMed  Google Scholar 

  45. Kuroki, R., Kawakita, S., Nakamura, H., and Yutani, K. (1989) Entropic stabilization of a mutant human lysozyme by calcium binding. Proc. Natl. Acad. Sci. USA. 89, 6803–6807.

    Article  Google Scholar 

  46. Haezebrouck, P., Baestlier, A. D., Joniau, M., Van Dael, H., Rozenberg, S., and Hanssens, I. (1993) Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme. Protein Eng. 6, 643–649.

    Article  CAS  PubMed  Google Scholar 

  47. Anderson, P. J., Brooks, C. L., and Berliner, L. J. (1997) Functional identification of calcium binding residues in bovine α-lactalbumin. Biochemistry 36, 11,648–11,654.

    Article  CAS  PubMed  Google Scholar 

  48. Kuroki, R. and Yutani, K. (1998) Structural and thermodynamic responses of mutations at a Ca2+binding site engineered into human lysozyme. J. Biol. Chem. 273, 34,310–34,315,.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kitaku, Sapporo, Japan

    Katsutoshi Nitta

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  1. Katsutoshi Nitta
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Editors and Affiliations

  1. Department of Biological Sciences, University of Calgary, Calgary, AB, Canada

    Hans J. Vogel

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© 2002 Humana Press Inc.

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Nitta, K. (2002). α-Lactalbumin and (Calcium-Binding) Lysozyme. In: Vogel, H.J. (eds) Calcium-Binding Protein Protocols. Methods in Molecular Biology™, vol 172. Humana Press. https://doi.org/10.1385/1-59259-183-3:211

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  • DOI: https://doi.org/10.1385/1-59259-183-3:211

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-688-8

  • Online ISBN: 978-1-59259-183-1

  • eBook Packages: Springer Protocols

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