FACTORS IN THE PREVENTION OF TOXIC LIVER INJURY

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.