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Abstract
Transforming growth factor β (TGF-β) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-β in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a mink lung epithelial cell line. Most of the TGF-β bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-β concentration was 4·3 ± 0·8 ng and 3·7 ± 0·7 ng TGF-β per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6·5 was used to concentrate latent whey-derived TGF-β, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-β. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-β with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-β from both high molecular mass peaks. Whey-derived TGF-β was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-β2, which accounted for over 85% of the TGF-β bioactivity in whey. All TGF-β activity in whey could be neutralised by a monoclonal antibody directed against TGF-β1, -β2 and -β3. The results suggest that the majority of TGF-β in bovine milk is present in a small latent complex.
Journal of Endocrinology (1996) 151, 77–86
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Abstract
Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80–100 kDa. In contrast, acid gelfiltration chromatography resolved two peaks of cell growth activity. A peak at 15–25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bioactivity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15–25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor β (TGF-β), and all inhibitory activity for Mv1Lu cells was immuno-neutralised by an antibody against TGF-β. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-β and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-β; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-β account for the 15–25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.
Journal of Endocrinology (1997) 154, 45–55
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Abstract
GH and IGF-I plasma concentrations were measured in lambs during an acute phase response induced by an intrathoracic injection of yeast. The acute phase response was indicated by reduced feed intake, weight loss and an increase in plasma concentrations of the acute phase protein haptoglobin. Intensive blood sampling on day 1 revealed elevated basal concentrations of GH in the yeast-injected group compared with concentrations in pair weight and ad libitum fed control lambs. This suggests that at the beginning of an acute phase response there is an increase in either GH secretion or the half life of GH. No evidence of a specific GH-binding protein in sheep plasma could be detected. IGF-I concentrations in the yeastinjected group remained constant for 3 days then increased to a peak level at day 6. In contrast, plasma IGF-I concentrations were depressed from days 3 to 6 in the pair weight control group and they were unchanged in the ad libitum fed controls. When the IGF-I concentrations were elevated in the yeast-injected group, this group had a higher daily weight gain despite their lower feed intake compared with the ad libitum fed controls. These results suggest that IGF-I may be associated with the increase in weight in the late stage of an acute phase response during recovery from an infection or injury. Day 1 GH peak amplitude concentrations in the yeast-injected lambs were negatively correlated with IGF-I concentrations on the following 2 days yet in the pair weight lambs the correlation was in a positive direction suggesting that the relationship between GH and IGF-I is different between animals that lose weight during an acute phase response and animals that lose weight because of feed restriction.
Journal of Endocrinology (1995) 144, 243–250
