During the purification of skeletal growth factor/insulin-like growth factor-II and transforming growth factor-beta (TGF beta) from EDTA extracts of bovine bone matrix significant mitogenic activity for cultured osteoblast (Ob)-like cells eluted in fractions that contained a nearly homogeneous peptide with a mol wt of about 14,000. This peptide has been purified to apparent homogeneity and identified as bovine beta 2-microglobulin (beta 2M) by amino-terminal amino acid sequencing. During the final purification of beta 2M by CN reverse phase HPLC the mitogenic activity for bone cells separated from the beta 2M protein peak. In spite of this the apparently homogeneous beta 2M preparation retained some mitogenic activity. The ED50 of the bone-derived beta 2M (4,890 +/- 462 ng/ml) was several orders of magnitude (2 x 10(3) to 1.4 x 10(5) times) greater than the ED50 of simultaneously assayed purified growth factors and was no different from the ED50 of the crude EDTA matrix extract (3,350 +/- 890 ng/ml). The beta 2M accounted for less than 0.002% of the total mitogenic activity for Ob-like cells present in the extracted matrix proteins. The following lines of evidence suggest that the mitogenic activity of bone matrix-derived beta 2M (BMD-beta 2M) is due to contamination of the BMD-beta 2M with TGF beta rather than an intrinsic property of beta 2M: 1) the coelution of TGF beta through four successive purification procedures and purification of TGF beta from adjoining fractions from C4 reverse phase HPLC; 2) the abolition of biological activity of BMD-beta 2M and TGF beta with reducing agents; 3) the lack of additive stimulation of [3H] methylthymidine incorporation into bone cells when beta 2M was added to maximally active concentrations of purified TGF beta; 4) the reduction of mitogenic activity when the BMD-beta 2M was incubated with anti-TGF beta; and 5) the inhibition of mink lung epithelial proliferation by the beta 2M preparation. Based on these findings we conclude that although beta 2M is present in bovine bone matrix extracts, it is not a mitogen for Ob-like cells.
Amino Acid Sequence Animals