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Human bone cells contain a fluoride sensitive acid phosphatase: evidence that this enzyme functions at neutral pH as
1Department of Medicine, Loma Linda University, CA.
Abstract:
Fluoride is a potent therapeutic agent that increases spinal bone density in osteoporotic subjects. Based on work with animal cells previously, we proposed fluoride acts by inhibiting phosphotyrosyl protein phosphatase (EC 3.1.3.48) activity in bone cells. The presence of fluoride sensitive acid phosphatase (EC 3.1.3.2) activity was characterized in extracts of cultured human bone cells. Crude extracts contained acid phosphatase activity that was inhibited by fluoride with an apparent Ki of 12 mumol/L. The activity was investigated further by separating the acid phosphatase isoenzymes using CM Sepharose chromatography and a gradient of acetate pH 4.8-6.5. The major peak of activity recovered from CM Sepharose chromatography was characterized for stability, Km and inhibition by fluoride. The enzyme was sensitive to inhibition by tartrate, had a high affinity for paranitrophenylphosphate (apparent Km = 0.158 mupmol/L) and an apparent pH optimum of 4.8. Fluoride was a strong competitive inhibitor with an apparent Ki of 12.4 mumol/L. The column fractions containing the acid phosphatase were tested further for phosphotyrosyl protein phosphatase activity using [32P]labeled phosphotyrosyl histone as the substrate. Release of [32P]phosphate from this substrate at pH 7.0 was proportional to enzyme concentration and incubation time, demonstrating the presence of phosphotyrosyl protein phosphatase activity. The phosphotyrosyl protein phosphatase activity was inhibited by fluoride and had a pH optimum of approximately 7. These observations indicate that human osteoblasts contain a fluoride-sensitive phosphotyrosyl protein phosphatase. Thus, these results are consistent with the hypothesis that fluoride stimulates human bone cell proliferation by inhibiting the action of phosphotyrosyl protein phosphatase, thereby increasing the level of phosphorylated tyrosine residues which are known to play a role in increasing cell proliferation.