Our objective in the proposed research is to study biochemical and immunological properties of enzymes involved in heme synthesis using recently developed techniques for enzyme purification and immunological methods. We plan to characterize the molecular properties of Delta-aminolevulinic acid (ALA) dehydratase, prophobilinogen (PBG) deaminase, and uroporphyrinogen decarboxylase in normal and trnsformed cells of erythroid origin and in cells derived from patients with inherited porphyrias. These enzymes will be purified from normal human erythrocytes and from mouse spleen or liver. The purified enzymes from mouse tissues will be used to prepare monospecific polyclonal antibodies in rabbits. The purified enzymes from human erythrocytes will be used to prepare monoclonal antibodies using the mouse hybridoma technique. Using rabbit antibodies directed to mouse enzymes, we plan to examine induction of enzyme proteins in mouse Friend erythroleukemia cells during erythroid differentiation induced by chemical treatment in culture. Characterization of heme pathway enzymes in human K562 erythroleukemia cells will be carried out using mouse monoclonal antibodies directed to human heme pathway enzymes purified from normal adult erythrocytes. By the use of monoclonal antibodies against human ALA dehydratase, the amount of the enzyme protein will be quantitatively examined in erythrocytes in patients with hereditary tyrosinemia. Monoclonal antibodies against human PBG deaminase will be utilized for the characterization of multiple forms of PBG deaminase in erythrocytes; possible molecular differences of PBG deaminase in cross-reactive immunological material (CRIM) positive individuals with the gene defect of acute intermittent porphyria. Examination of CRIM status of deficient uroporphyrinogen decarboxylse activityin erythrocytes from patients with porphyria cutanea tarda (PCT), particularly familial PCT (heterozygous and homozygous subjects for the gene defect), will be carried out using monoclonal antibodies directed to the normal enzyme. Information obtained through these studies should lead to a better understanding of the characteristic properties and mode of regulation of these heme pathway enzymes in normal erythrocytes, in transformed erythroid cells, and in erythrocytes from in individuals with inherited porphyric gene defects.
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