Chemistry Reference
In-Depth Information
The microsomal fraction consists mainly of vesicles (microsomes) derived
from the endoplasmic reticulum (smooth and rough). It contains cytochrome
P450 and NADPH/cytochrome P450 reductase (collectively the microsomal
monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases,
glucuronyl transferases, and other enzymes that metabolize xenobiotics. The
105,000 g supernatant contains soluble enzymes such as glutathione-
S
-trans-
ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant con-
tains all of the types of enzyme listed earlier.
Microsomes are widely used to study the metabolism of xenobiotics.
Enzymes can be chararacterized on the basis of their requirement for cofac-
tors (e.g., NADPH, UDPGA), and their response to inhibitors. Kinetic studies
can be carried out, and kinetic constants determined. They are very useful in
studies of comparative metabolism, where many species not available for in
vivo experiment can be compared with widely investigated laboratory species
such as rats, mice, feral pigeon, Japanese quail, and rainbow trout.
form an acetyl cysteine conjugate (also referred to as a mercapturic acid conjugate).
Mercapturic acid conjugates are often the main excreted forms following glutathi-
one conjugation in mammals. However, cysteine conjugates are also excreted and,
in insects, unchanged glutathione conjugates have been reported as the principal
excreted forms. A contrasting type of glutathione conjugation involves attack upon
epoxides. The epoxide ring is opened, a GS link is attached to one carbon, and OH
is attached to the other. The net effect is addition across the epoxide bond without
substitution. This latter type of conjugation can be critical in removing newly gener-
ated mutagenic epoxides such as benzo[
a
]pyrene 7,8-diol 9,10 epoxide (Figure 2.14)
before they can cause cellular damage by binding to DNA.
Glutathione-
S
-transferases are known to exist in a number of isoforms. These
are homo- or heterodimers, built from subunits of 22-28 kD. In rat, three classes of
isoforms are known, built on subunits numbered 1-7.
class
constitution
Alpha
1:1, 1:2, and 2:2
Mu
3:3, 3:4, 4:4, and 6:6
Pi
7:7
There is less than 30% sequence homology between the different classes. Although
glutathione-
S
-transferases are predominantly cytosolic enzymes, one microsomal
form is known, which exists as a trimer of molecular mass 51 kD. Some glutathi-
one conjugates are unstable and are better regarded as intermediates in the pro-
cess of biotransformation than as stable conjugates for excretion. Thus, with certain
organohalogen compounds, conjugation with glutathione involves dehydrohalogena-
tion before the conjugate breaks down to release a dehalogenated metabolite. One
example is the conjugation of dichloromethane. The conjugate is hydrolyzed, and
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