The proposed work investigates the distribution of heme within hepatocytes. We are engaged in purifying and characterizing cytosolic proteins of rat liver which bind heme and porphyrins. It is our aim to develop experimental systems in which we can study intracellular protein mediation both of heme transfer from the mitochondria to 1) the cytosol, 2) microsomes and 3) peroxisomes, and of its incorporation into the corresponding heme-containing enzymes (cytosolic) tryptophan pyrrolase, the (microsomal) cytochrome P450 isozymes and (peroxisomal) catalase. Parallel to these studies with subcellular organelles it is planned to continue our investigations using liposomes as model membranes for determinations of heme transfer kinetics in order to postulate likely mechanisms for the efflux of heme to cytosol and into subcellular organelles. In addition we will search for membrane proteins of subcellular organelles possibly facilitating the movement of heme into and from organelles. We will examine the association of heme and heme-binding cytosolic proteins putatively functioning as heme-carriers with these membranes and membrane proteins. Once these aspects of intracellular heme transport under normal conditions are defined, we will examine the same parameters under the influence of altered iron and porphyrin metabolism. Decreased hepatic heme levels will be produced by: 1) inhibiting heme synthesis or limiting iron supply; 2) blocking enzymatic steps of heme synthesis with various compounds; and 3) increasing the cellular demand by inducing cytochrome P450 isozymes. The results of the proposed work should provide much needed information on the physiology of intracellular heme transport and distribution, and should enhance our understanding of how aberrations of iron and porphyrin metabolism affect these processes. In addition, the research should provide information clinically useful in the treatment of porphyrias and also in the development of therapies involving porphyrin localization and photochemical mechanisms.
Showing the most recent 10 out of 24 publications
