The characterization of protein components produced from bone tissues with fracture healing was investigated. Weanling rats were sacrificed between 1 and 7 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that many protein molecules were released from the diaphyseal tissues with fracture healing. Especially, a protein molecule of approximately 66 kDa was markedly increased by fracture healing. This protein molecule was significantly increased, when the diaphyseal tissues with fracture healing were cultured in the presence of zinc acexamate (10-6-10-4 M). Zinc acexamate (10-4 M)-induced increase in medium 66 kDa protein molecule was significantly inhibited in the presence of actinomycin D (10-7 M) or cycloheximide (10-6 M). The zinc effect was completely blocked in the presence of PD98059 (10-5 M), an inhibitor of MAPK kinase, or staurosporine (10-6 M), an inhibitor of protein kinase C. The medium 66 kDa protein molecule was significantly elevated in the presence of parathyroid hormone (1-34) (10-7 M), insulin-like growth factor-I (10-8 M) or transforming growth factor-β (10-11 M), while 17β-estradiol (10-9 M) did not have an effect. The effect of these bone-stimulating factors was equal to the zinc effect. Zinc did not significantly enhance the effect of insulin-like growth factor-I in increasing medium 66 kDa protein molecule. The present study demonstrates that fracture healing increases production of the ≈66 kDa protein molecule which is a major component produced from femoral-diaphyseal tissues of weanling rats, and that this elevation is enhanced by zinc treatment.

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