Wergedal J E, Lau K H
Department of Medicine, Loma Linda University, CA.
Clin Biochem. 1992 Feb;25(1):47-53. doi: 10.1016/0009-9120(92)80045-i.
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.
氟化物是一种有效的治疗剂，可增加骨质疏松症患者的脊柱骨密度。基于之前对动物细胞的研究工作，我们提出氟化物通过抑制骨细胞中的磷酸酪氨酸蛋白磷酸酶（EC 3.1.3.48）活性来发挥作用。在培养的人骨细胞提取物中对氟化物敏感的酸性磷酸酶（EC 3.1.3.2）活性进行了表征。粗提取物中含有酸性磷酸酶活性，该活性被氟化物抑制，表观抑制常数（Ki）为12 μmol/L。通过使用CM Sepharose色谱法和pH 4.8 - 6.5的醋酸盐梯度分离酸性磷酸酶同工酶，对该活性进行了进一步研究。从CM Sepharose色谱法中回收的主要活性峰对稳定性、米氏常数（Km）和氟化物抑制作用进行了表征。该酶对酒石酸盐抑制敏感，对对硝基苯磷酸酯具有高亲和力（表观Km = 0.158 μmol/L），表观最适pH为4.8。氟化物是一种强竞争性抑制剂，表观Ki为12.4 μmol/L。使用[32P]标记的磷酸酪氨酸组蛋白作为底物，对含有酸性磷酸酶的柱级分进一步检测磷酸酪氨酸蛋白磷酸酶活性。在pH 7.0时从该底物释放的[32P]磷酸盐与酶浓度和孵育时间成正比，表明存在磷酸酪氨酸蛋白磷酸酶活性。磷酸酪氨酸蛋白磷酸酶活性被氟化物抑制，最适pH约为7。这些观察结果表明人成骨细胞含有一种氟化物敏感的磷酸酪氨酸蛋白磷酸酶。因此，这些结果与以下假设一致，即氟化物通过抑制磷酸酪氨酸蛋白磷酸酶的作用来刺激人骨细胞增殖，从而增加已知在增加细胞增殖中起作用的磷酸化酪氨酸残基的水平。
