This proposal seeks continued funding to study a group of unusual, iron-containing acid phosphatase - the so called tartrate-resistant acid phosphatase (or TR-AP's). The best studied of this family of enzymes is uteroferrin (UF), a secreted, progesterone-induced TR- AP which has been implicated in transplacental iron transport in pigs and whose novel bi-iron center and purple colors has excited much recent attention. The first objective is to test the hypothesis that intracellular TR-AP's which are believed to be located in the lysosome-endosome compartment, are involved as intermediaries in iron metabolism of the erytholeukemic cell line, K562. Related to this study will be a purification of the TR-AP, its characterization and a description of its intracellular localization. The manner in which Fe availability and the state of differentiation of the cells regulate TR-AP levels will also be examined, and the flow of 59-iron through the intracellular pool of the enzyme followed under a variety of conditions. Objective 2 is crystallize UF and define its structure by x-ray diffraction techniques. Such information is an important prerequisite for any rational series of experiments designed to study Uf targeting and processing by the technique of site-directed mutagenesis. The experiments are also expected to define the bi-iron center on Uf more clearly and possibly the region on the molecule that makes it a substrate for the Golgi enzyme N-acetyl D-glucosamine 1-phosphate transferase. Objective 3 is to determine whether Uf is secreted as a result of targeting information in its primary sequence. Here we shall compare enzymatic and other properties of Uf (which is secreted) and pig spleen TR-AP (which is intracellular and probably lysosomal). The targeting of both proteins will be compared in cell lines transfected with vectors containing cDNA's. Provided crystallographic information becomes available, the Uf cDNA will be modified by site directed mutagenesis to determined whether the movement of the protein within the cell can be redirected. Objective 4 is to determine the basis of TR-AP elevation in certain leukemias. Here our aim will be to complete the cloning of the cDNA to human placental TR-AP and to use this cDNA as a probe a) to determine whether the mRNA levels, mRNA transcription rates and mRNA stability area responsible for elevated TR-AP levels, b) to determine whether there are specific chromosomal rearrangements of the TR-AP gene in hairy cell and other leukemias and to define the specific chromosomal localization of the human TR-AP gene(s) in normal cells.
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