, Volume 167, Issue 3–4, pp 134–144 | Cite as

Identification of human milk α-lactalbumin as a cell growth inhibitor

  • M. P. Thompson
  • H. M. FarrellJr.
  • Sanjeeva Mohanam
  • Sue Liu
  • W. R. Kidwell
  • M. P. Bansal
  • R. G. Cook
  • D. Medina
  • Claire E. Kotts
  • Mozeena Bano


A growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of α-lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human α-lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and α-lactalbumin are identical proteins. Consistent with these results, α-lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human α-lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation α-lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.


Milk protein Mammary cells Cell growth Inhibition 



mammary inhibitory activity


mammary derived growth inhibitor


alpha lactalbumin


human α-lactalbumin


normal rat kidney


improved minimal essential medium


Dulbecco's modified Eagles medium


fetal calf serum


epidermal growth factor


transforming growth factor β


cyanogen bromide


sodium dodecyl sulfate




non-denaturing polyacrylamide gel electrophoresis


trichloroacetic acid


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akers RM (1990) Lactation physiology: a ruminant animal perspective. Protoplasma 159: 96–111Google Scholar
  2. —, McFadden TB, Beal WE, Guidry AJ, Farrell HM Jr (1986) Radioimmunoassay for measurement of bovine α-lactalbumin in serum, milk and tissue culture media. J Dairy Res 53: 419–429PubMedGoogle Scholar
  3. Aschaffenburg R, Fenna RE, Phillips DC, Smith SG, Buss DH, Jenness R, Thompson MP (1979) Crystallography of α-lactalbumin III. Crystals of baboon milk α-lactalbumin. J Mol Biol 127: 135–137PubMedGoogle Scholar
  4. Bano M, Salomon DS, Kidwell WR (1985) Purification of mammary-derived growth factor from human milk and mammary tumours. J Biol Chem 260: 5745–5752PubMedGoogle Scholar
  5. Bansal MP, Cook RG, Danielson KG, Medina D (1989) A 14 kilodalton selinium-biriding protein in mouse liver is fatty acid binding protein. J Biol Chem 264: 13780–13784PubMedGoogle Scholar
  6. Berliner LJ, Meinholtz DC, Hirai Y, Musci G, Thompson MP (1991) Functional implications resulting from disruption of the calciumbinding loop in α-lactalbumin. J Dairy Sci 74: 2394–2402PubMedGoogle Scholar
  7. Böhmer FD, Lehmann W, Noll F, Samtleben R, Langen P, Grosse R (1985) Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland. Biochim Biophys Acta 846: 145–154PubMedGoogle Scholar
  8. —, Kraft R, Otto A, Wernstedt V, Hellmann U, Kurtz A, Muller T, Rhode K, Etzold W, Lehmann W, Langen P, Grosse R (1978) Identification of a polypeptide growth inhibitor from bovine mammary gland. J Biol Chem 262: 15137–15143Google Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of dye binding. Anal Biochem 72: 248–251PubMedGoogle Scholar
  10. Brew K, Castellino FJ, Vanaman TD, Hill RL (1970) The complete amino acid sequence of bovine α-lactalbumin. J Biol Chem 245: 4570–4582PubMedGoogle Scholar
  11. Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecylsulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102–1106PubMedGoogle Scholar
  12. Cornish-Bowden A (1983) Relating proteins by amino acid composition. In: Hirs CHW, Timasheff Sn (eds) Methods in enzymology, vol 91. Academic Press, New York, p 60Google Scholar
  13. DeGeyter C, Cooper TG, DeGeyter M, Nieschlag E (1989) Effects of bovine mammary α-lactalbumin on hyperactivation and sperm-zona pellucida binding of mouse spermatazoa. Gamete Res 24: 415–426PubMedGoogle Scholar
  14. Doyen N, Lapresle C (1979) Partial non-cleavage by cyanogen bromide of a methionine-cystine bond from human serum albumin and bovine α-lactalbumin. Biochem J 177: 251–254PubMedGoogle Scholar
  15. Ebner KE, Denton WL, Brodbeck U (1966) The substitution of α-lactalbumin for the B protein of lactose synthetase. Biochem Biophys Res Comm 24: 232–236PubMedGoogle Scholar
  16. Farrell HM Jr (1988) Physical equilibria: proteins In: Wong N (ed) Fundamentals of dairy chemistry. Van Nostrand Reinhold, New York, pp 461–510Google Scholar
  17. —, Thompson MP (1990) β-Lactoglobulin and α-lactalbumin as potential modulators of mammary cellular activity. A Ca2+ responsive model system using acid phosphoprotein phosphatases. Protoplasma 159: 157–167Google Scholar
  18. —, Deeney JT, Hild EK, Kumosinski TF (1990) Stopped flow and steady state kinetic studies of the effects of metabolites on NADP+: isocitrate dehydrogenase. J Biol Chem 265: 17637–17643PubMedGoogle Scholar
  19. —, Kumosinski TF, Pulaski P, Thompson MP (1988) Calcium-induced associations of the caseins; a thermodynamic linkage approach to precipitation and resolubilization. Arch Biochem Biophys 265: 146–158PubMedGoogle Scholar
  20. Fenna RE (1982) Crystallization of human α-lactalbumin. J Mol Biol 161: 211–215PubMedGoogle Scholar
  21. Fling SP, Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using high molarity Tris buffer without urea. Anal Biochem 155: 83–88PubMedGoogle Scholar
  22. Herrmann I, Grosse R (1986) Characterization of membrane-associated growth inhibitor activity for Ehrlich acites mammary carcinoma cells. Biomed Biochim Acta 45: 447–457PubMedGoogle Scholar
  23. Humphreys-Beher MG, Schneyer CA, Zelles T (1987) α-Lactalbumin acts as a bimodal regulator of rat parotid acinar cell growth. Biochem Biophys Res Comm 147: 174–181PubMedGoogle Scholar
  24. Kidwell WR, Bano M, Salomon DS (1984) In: Barnes DW, Sibasku DA, Sato GA (eds) Cell culture methods for molecular and cell biology, vol 2. Alan R Liss, New York, pp 105–125Google Scholar
  25. Klagsbrun M (1978) Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts. Proc Natl Acad Sci USA 75: 5057–5061PubMedGoogle Scholar
  26. Klagsbrun M, Shing Y (1984) Growth-promoting factors in human and bovine milk. In: Guroff G (eds) Growth and maturation factors, vol 2. Wiley, New York, pp 161–192Google Scholar
  27. Kurtz A, Vogel F, Funa K, Heldin C-H, Grosse R (1990) Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue. J Cell Biol 110: 1779–1789PubMedGoogle Scholar
  28. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedGoogle Scholar
  29. Leung CT, Maleeff BE, Farrell HM Jr (1989) Subcellular and ultrastructural localization of alkaline phosphatase in lactating rat mammary glands. J Dairy Sci 72: 2495–2509PubMedGoogle Scholar
  30. Lucas C, Bald LN, Fendly BM, Mora-Worms M, Figari IS, Patzer EJ, Palladino MA (1990) The autocrine production of transforming growth factor-β1 during lymphocyte activation. J Immunol 145: 1415–1422PubMedGoogle Scholar
  31. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035–10038PubMedGoogle Scholar
  32. Murakami K, Andree PJ, Berliner LJ (1982) Metal ion binding to α-lactalbumin species. Biochemistry 21: 5488–5494PubMedGoogle Scholar
  33. Musci G, Berliner LJ (1985) Physiological roles of zinc and calcium binding to α-lactalbumin in lactose biosynthesis. Biochemistry 24: 6945–6948PubMedGoogle Scholar
  34. Palmiter RD (1969) Properties of lactose synthetase from mouse mammary gland: role of a proposed third component. Biochim Biophys Acta 178: 35–46PubMedGoogle Scholar
  35. Quarfoth GJ, Jenness R (1975) Isolation, composition and functional properties of α-lactalbumin from several species. Biochim Biophys Acta 379: 476–487PubMedGoogle Scholar
  36. Richter A, Sanford KK, Evans VJ (1972) Influence of oxygen and culture media on plating efficiency of some mammelian tissue cells. J Natl Cancer Inst 49: 1705–1712PubMedGoogle Scholar
  37. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB (1985) Type β transforming growth factor: a bifunctional regulator of cell growth. Proc Natl Acad Sci USA 82: 119–123PubMedGoogle Scholar
  38. Salomon DS, Zwiebel JA, Bano M, Losonczy I, Fehnel P, Kidwell WR (1984) Presence of transforming growth factors in human breast cancer cells. Cancer Res 44: 4069–4077PubMedGoogle Scholar
  39. Shing YW, Klagsbrun M (1984) Human and bovine milks contain different sets of growth factors. Endocrinology 115: 273–282PubMedGoogle Scholar
  40. Simickova M, Lang BA, Semotan K, Kocent A (1987) A non-competitive enzyme immunoassay of human α-lactalbumin in biological fluids and tissue extracts. Clin Chim Acta 163: 257–265PubMedGoogle Scholar
  41. Stampfer M, Hallowes RC, Hackett AJ (1980) Growth of normal human mammary cells in culture. In Vitro 16: 415–425PubMedGoogle Scholar
  42. Stuart DI, Acharya KR, Walker NPC, Smith SG, Lewis M, Phillips DC (1986) α-Lactalbumin possesses a novel calcium binding loop. Nature 324: 84–87PubMedGoogle Scholar
  43. Thompson MP, Brower DP (1988) The method of Aschaffenburg and Drewry for the crystallization of β-lactoglobulin and α-lactalbumin. J Dairy Sci 71: 1141–1146Google Scholar
  44. —, Piazza GJ, Brower DP, Farrell HM Jr (1989) Purification and characterization of calmodulins fromPapaver somnifera andEuphorbia lathyris. Plant Physiol 89: 501–505Google Scholar
  45. —, Groves ML, Brower DP, Farrell HM Jr, Jenness R, Kotts CE (1988) The calcium-dependent electrophoretic shift of α-lactalbumin, the modifier protein of galactosyl transferase. Biochem Biophys Res Comm 157: 944–948PubMedGoogle Scholar
  46. —, Piazza GJ, Brower DP, Bingham EW, Farrell HM Jr (1987) Isolation and characterization of bovine mammary calmodulin. J Dairy Sci 70: 1551–1556PubMedGoogle Scholar
  47. Wang JL, Hsu YM (1986) Negative regulators of cell growth. Trends Biochem Sci 11: 24–26Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • M. P. Thompson
    • 8
  • H. M. FarrellJr.
    • 8
  • Sanjeeva Mohanam
    • 1
  • Sue Liu
    • 2
  • W. R. Kidwell
    • 3
  • M. P. Bansal
    • 4
  • R. G. Cook
    • 5
  • D. Medina
    • 4
  • Claire E. Kotts
    • 6
  • Mozeena Bano
    • 7
  1. 1.Cancer Biology Division, School of Biological SicencesMadurai Kamaraj UniversityMaduraiIndia
  2. 2.Surgery Branch, National Cancer InstituteNIHBethesda
  3. 3.Cellco Advanced Bioreactors, Inc.Kensington
  4. 4.Department of Cell BiologyBaylor College of MedicineHouston
  5. 5.Howard Hughes Medical Institute and Department of Microbiology and ImmunologyBaylor College of MedicineHouston
  6. 6.Department of Medicinal and Analytical ChemistryGenentech Inc.South San Francisco
  7. 7.Lombardi Cancer Research CenterGeorgetown UniversityWashington, D.C.
  8. 8.ARS, Eastern Regional Research CenterUSDAPhiladelphiaUSA

Personalised recommendations