The glutathione S-transferases (GSH-Tases) are a family of enzymes that are present in high concentration within the hepatic cytosol. The GSH-Tases are important enzymes for the detoxification of a large number of reactive molecules. These same proteins are also thought to function as transport proteins for a variety of non-polar molecules (bilirubin, bile acids, heme) that they do not metabolize. The enzymatic and transport functions are often at variance with each other as the nonsubstrate ligands can be inhibitors or enzymatic activity. It is uncertain, therefore, how these proteins carry out these two competing functions. This is an important question because high intracellular concentrations of bilirubin and bile acids, as seen in patients with liver disease, could reduce the activity of the GSH- Tases to a level where the liver would become suspectible to injury by drugs or toxins normally metabolized by the GSH-Tases. In this proposal the effect of the accumulation of bilirubin and bile acids within cultured hepatocytes on the ability of the GSH- Tases to metabolize drugs will be determined. The products of GSH-Tase catalyzed reactions are also potent inhibitors of these enzymes. In addition, some of the glutathione conjugates are themselves toxic and their rapid biliary excretion is essential both to prevent liver injury and inhibition of the GSH-Tases. In this proposal the properties of the carrier in the canalicular membrane that excretes these glutathione conjugates will be investigated in cultured hepatocytes. The polypeptides involved in biliary excretion will be identified in purified canalicular membranes by use of a photoaffinity label. The size of the carrier will be determined by radiation inactivation. Many of the drug metabolizing enzymes in the endoplasmic reticulum (ER) contain heme (cytochromes). Heme must move from its site of synthesis in the mitochondria to the ER to form the active cytochromes from the respective apoproteins. The GSH-Tases may facilitate this movement of heme and a failure of the GSH-Tases to perform this important function could lead to significant decreases in the levels of the cytochromes in the ER. The ability of and mechanism by which the GSH-Tases transport heme from the mitochondria to the apocytochromes will be investigated using purified GSH-Tases, apocytochrome b5 and mitochondria as the source of heme. The GSH-Tases are dimeric proteins and there has been interest in how subunit interactions effect function. This problem will be studied by the use of radiation inactivation to destroy one of the subunits while leaving the second intact.
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