A fluoride-sensitive acid phosphatase activity is clearly present in osteoblast-line cells, however, its function is unknown. Based on the preliminary findings regarding this enzyme which are described in this application, the following hypotheses have been developed: (1) F-sensitive acid phosphatase is unique to osteoblast-line cells, (2) this enzyme acts as a neutral pH phosphotyrosyl phosphatase in proliferating bone cells, (3) this enzyme is, at least in part, a significant determinant of phosphotyrosyl protein levels in these cells, and (4) inhibition of this enzyme by specific inhibitors will serve to increase tyrosyl phosphorylation and stimulate bone cell proliferation. In this application, the first two hypotheses will be tested by purifying and characterizing this enzyme (and its neutral pH phosphotyrosyl phosphatase activity) from chick osteoblast-line cells, and determining its primary sequence. Specific antibodies against the purified enzyme will be developed and characterized, and they will be used in studies to test hypotheses 3 and 4. Because it is essential to identify physiologic substrates for an enzyme before its function can be tested, cellular substrates of this F-sensitive acid phosphatase will be identified by determining those cellular proteins whose tyrosyl phosphorylation are stimulated by (1) inhibitors of the enzyme (ie., F, vanadate, and molybdate) and (2) by specific inhibitory antibodies. [The P-tyr proteins will be isolated with anti-Tyr(P) antibodies.] The long term goal of these studies is to test the model that inhibition of phosphotyrosyl phosphatase can lead to cell proliferation in general (i.e., hypothesis 4). If my hypotheses are correct, the significance of these proposed studies will be two-fold: (1) They would provide important information on a key regulatory process (i.e., cell proliferation) in bone. More importantly, this information I derive about this system of P-tyr phosphorylation may be applicable to all renewing cell populations, not merely bone cells. (2) They will advance our understanding of the molecular mechanism of action of F, which is an effective therapeutic agent in the treatment of spinal osteoporosis. Knowledge of the mechanism of action of F could provide background information for development of new therapeutic treatments, similar in mechanism to F but more effective and less toxic.
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