Fetuin and alpha‐2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes

A previously described chondrocyte alkaline phosphatase induction factor (CAP‐IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS‐PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye‐ligand affinity (Affi‐Gel Blue and Reactive Green‐19 agarose) and hybroxyapatite column chromatographies. As determined by immunoprecipitation of [ 35 S]methionine‐labeled cellular proteins after 3 day treatment, this highly purified CAP‐IF increases the level of AP and certain other membrane proteins 2‐ to 3‐fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N‐terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)‐inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP‐IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn 2+ did not reactivate the EDTA‐treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8‐fold during 3 day exposure. Because of the very high homology between fetuin and the A‐chain of α 2 ‐HS glycoprotein, we also tested and found that α 2 HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP‐inducing activity resides in a labile, cystatin/Zn 2+ ‐binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.