FUNCTION OF PURPLE ACID PHOSPHATASE IN DEVELOPING BONE

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The overall goals of our research on purple, tartrate-resistant acid phosphatase (E2) in osteoclasts are to delineate its function and determine if it serves a role in protecting the cell, and subsequent bone remodeling, against damage by iron toxicity and free radicals. This proposal covers initial steps of an in vitro study designed to determine if E2: (1) can function inside or outside the cell, (2) is under different controls than are lysosomal hydrolases, and (3) scavenges iron and orthophosphate (orthophosphate). We have determined that long bones of rat pups contain much higher levels of E2 than of tartrate-sensitive (E1) activity; we also have purified E2 and immunocytochemically localized it to osteoclast vesicles. E2 is the only known acid phosphatase with a metal (i.e. iron) moiety. One-iron E2 may function similarly to transferrin as a sink for free iron. Two-iron E2 may function in two ways: (1) The redox potential of the second iron can catalyze redox reactions of lower potential, and (2) if the second iron is reduced, then E2 becomes active as a phosphatase. However, the latter two possible functions would not occur in resorption lacunae where the high orthophosphate content would maintain E2 catalytically inactive. Therefore, bone E2 may function as a sink for free iron, which would be kept immobilized by orthophosphate bound to the iron. Free iron released during bone resorption must be sequestered because the acidic and aerobic milieu of the lacunae would favor formation of free radicals. The secretion of E2 from isolated osteoclasts, and the effects of exogenous iron and orthophosphate will be determined with a reverse hemolytic plaque assay, which can show secretion by single cells. These effects on secretion will also be quantitated with immunoelectrophoresis and a fluorescent enzyme assay that is 1000x more sensitive than standard assays for E2. Levels of E2 inside and outside the cell will be compared to those of two known lysosomal hydrolases, E1 and 2- glucuronidase, to determine possible differences in behavior between the enzymes. Assays for the latter two enzymes will use the same fluorescent assay as for E2, and means will be used to distinguish between E1 and E2. If the results support secretion of E2 especially in response to exogenous iron and orthophosphate, it will be determined if E2 can scavenge these ions. Activation of E2 phosphatase activity by the addition of the second iron atom to E2, and its subsequent reduction with ascorbate, will serve as the basis of an assay for iron scavenging; inactivation of E2 with orthophosphate will form the basis of an assay for the scavenging of orthophosphate.