Transforming growth factor beta in bovine milk: Concentration, stability and molecular mass forms

Transforming growth factor beta (TGF-beta) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-beta in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a mink lung epithelial cell line. Most of the TGF-beta 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-beta concentration was 4.3 +/- 0.8 ng and 3.7 +/- 0.7 ng TGF-beta per mi of milk and cheese whey respectively. Cation-exchange chromatography at pH 6.5 was used to concentrate latent whey-derived TGF-beta, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-beta. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-beta 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-beta from both high molecular mass peaks. Whey-derived TGF-beta was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-beta 2, which accounted for over 85% of the TGF-beta bioactivity in whey. All TGF-beta activity in whey could be neutralised by a monoclonal antibody directed against TGF-beta 1, -beta 2 and -beta 3. The results suggest that the majority of TGF-beta in bovine milk is present in a small latent complex.