(Received for publication, October 10, 1994)
From the
Alcohol:NAD oxidoreductase was found in the
peroxisomes of animal liver for the first time as follows. The
distribution of alcohol:NAD
oxidoreductase activity
with nonanol as substrate in the light mitochondrial fraction
(peroxisome-enriched fraction) of rat liver was examined by
centrifugation in a sucrose density gradient. Most of the enzyme
activity was localized in the mitochondria, with some activity in the
peroxisomes. The administration of clofibrate, a peroxisome
proliferator, to rats resulted in a marked increase of the enzyme
activity in the peroxisomes, but not in the mitochondria. The enzyme
was found to be located in the matrix of the peroxisomes. The evidence
was obtained that the enzyme differed from alcohol dehydrogenases and
alcohol oxidizing systems found previously. The enzyme activity was not
affected by pyrazole, an inhibitor of alcohol dehydrogenase and sodium
azide, an inhibitor of catalase. The enzyme was
NAD
-dependent and oxidized straight chain aliphatic
alcohols with a variety of carbon chains
(C
-C
), showing the maximum on nonanol. K
values toward these aliphatic alcohols
decreased with increasing chain length. The major reaction product was
identified as the carboxylic acid by using high performance liquid
chromatography.
Three separate enzymes have been reported to be involved in
conversion of ethanol to acetaldehyde in mammalian liver. 1) The most
widely known is the NAD-linked cytosolic alcohol
dehydrogenase (EC 1.1.1.1), which catalyzes the reversible
interconversion of a wide variety of alcohols and their corresponding
aldehydes(1, 2) . 2) A mixed function oxidase (EC
1.6.2.4, 1.14.14.1), known as the microsomal ethanol-oxidizing system,
catalyzes the NADPH and oxygen-dependent oxidation of short chain
alcohols to corresponding aldehydes(3) . 3) Catalase, which is
present in the peroxisomes, can also catalyze the oxidation of short
chain alcohols to corresponding aldehydes by its peroxidatic
activity(4) . The latter two enzymes are
NAD
-independent and cannot oxidize higher primary
alcohols(5) .
In the present study, we report that a fourth
enzyme that can oxidize aliphatic alcohols with a variety of carbon
chain (C-C
) to corresponding aldehydes
is present in the peroxisomes of rat liver. The enzyme is
NAD
-dependent and induced by the administration of
clofibrate, a peroxisome proliferator.
Alcohol:NAD oxidoreductase activities with
nonanol as substrate were determined in subcellular fractions from
normal and clofibrate-treated rat livers (Table 1).
Clofibrate treatment resulted in the increase of the specific activity of the enzyme only in the light mitochondrial fraction (peroxisome-enriched fraction) but not in other fractions (Table 1).
The result suggests that the enzyme is present in the peroxisomes of rat liver, because clofibrate treatment of rats is known to cause proliferation of the peroxisomes and increase of peroxisomal enzyme activities in liver(14) .
Fig. 1shows the representative
sedimentation profiles of the light mitochondrial fractions
(peroxisome-enriched fractions) from normal and clofibrate-treated rat
liver in a sucrose density gradient. The peroxisomes and mitochondria
were separated; the peroxisomes, marked by catalase, was only at a
density of about 1.25 g/ml, and the mitochondria, marked by glutamate
dehydrogenase, only at a density of about 1.18 g/ml (Fig. 1A). Compared with normal rats, liver from
clofibrate-treated rats showed a marked increase (about 10-fold) in the
peroxisomal -oxidation activity (palmitoyl-CoA-dependent
NAD
reduction) (Fig. 1B). These
results show that liver peroxisomes proliferated in clofibrate-treated
rats(15, 16) .
Figure 1:
Subcellular distribution of
alcohol:NAD oxidoreductase in normal and
clofibrate-treated rat livers. The light mitochondrial fractions were
prepared from livers of normal and clofibrate-treated rats and were
separately subjected to sucrose density gradient centrifugation as
described in the text. Fractions of 2.2 ml were collected from the
bottom of each tube. A, glutamate dehydrogenase (
) and
catalase (
) of normal rat liver; B,
palmitoyl-CoA-dependent NAD
reduction activities of
normal (
) and clofibrate-treated (
) rat liver; C,
alcohol:NAD
oxidoreductase activity of normal (
)
and clofibrate-treated (
) rat liver.
On the other hand, in normal rat,
most of alcohol:NAD oxidoreductase activity with
nonanol as substrate, was recovered in the mitochondria, with some
activity in the peroxisomes (Fig. 1C). However, the
treatment with clofibrate resulted in a marked increase (about 5-fold)
of the peroxisomal enzyme activity with no effect on the mitochondrial
one. These results clearly demonstrate that alcohol:NAD
oxidoreductase is located in the peroxisomes of rat liver.
To
examine intraperoxisomal localization of alcohol:NAD oxidoreductase activity with nonanol as substrate, the light
mitochondrial fraction was prepared from livers of clofibrate-treated
rats. The light mitochondrial fraction, containing 60 µg of
protein, was suspended in 20 mM glycylglycine, pH 7.5,
containing 0.25 M sucrose and various concentrations
(0-0.3 M) of KCl. After incubation at 4 °C for 15
min, each suspension was centrifuged at 12,500
g for
30 min and each resulting supernatant was assayed for unsedimentable
activity. KCl (up to 0.3 M) did not solubilize any the
catalase as the peroxisomal matrix marker or alcohol:NAD
oxidoreductase activity, showing that alcohol:NAD
oxidoreductase is not the peripheral membrane protein that is
simply associated with peroxisomes by ionogenic interaction (data not
shown).
Next, the peroxisomal fractions prepared from livers of
clofibrate-treated rats (Fig. 1C, fractions2-4) were combined and diluted with the same volume
of 0.01 M pyrophosphate buffer, pH 10.5, which is known to
break rat liver peroxisomes(17) . After being stored overnight
at 4 °C, about 6 ml of the suspension was subjected to sucrose
density gradient centrifugation as described previously (10) (Fig. 2). Catalase as the peroxisomal matrix marker
was completely solubilized and recovered in the soluble top fraction,
and NADH-cytochrome c reductase in the membrane is distributed
over a broad density range with a peak of about 1.17 g
ml
(10, 18) . Nearly all of the
alcohol:NAD
oxidoreductase activity was recovered in
the soluble top fraction. These results show that
alcohol:NAD
oxidoreductase is located in the
peroxisomal soluble matrix.
Figure 2:
Intraperoxisomal localization of
alcohol:NAD oxidoreductase. Peroxisomal fractions (fractions 2-4 in Fig. 1C), isolated on
a sucrose density gradient, were diluted with the same volume of 0.01 M pyrophosphate butter, pH 10.5, which is known to break rat
liver peroxisomes. After being stored overnight at 4 °C, the
suspension (about 6 ml) was layered on 28 ml of a sucrose gradient
(30-56%, w/w) solution and centrifuged at 132,000
g for 70 min. Fractions of 2.2 ml were collected from the bottom of
the tube. A, NADH-cytochrome c reductase (
) and
catalase (
); B, alcohol:NAD
oxidoreductase activity (
).
Fig. 3represents high
performance liquid chromatograms of the UV-derivatives of the reaction
products. The solubilized fractions of the peroxisomes in Fig. 2(fractions 14-16) were combined and used as
the peroxisomal alcohol:NAD oxidoreductase
preparation. The reaction system contained 100 µl (0.1 milliunit)
of the enzyme preparation in the standard assay mixture. After
incubation at 37 °C for 17 h, the products were extracted and
converted to the appropriate derivatives as described under
``Experimental Procedures''. The 4-nitrobenzyl derivatives of
nonanal and nonanoic acid as the standards were separated on an ODS
column; the 4-nitrobenzyl oxime, corresponding 30 nmol of nonanal, was
at a retention time of about 12.7 min and the 4-nitrobenzyl ester,
corresponding 10 nmol of nonanoic acid, was at a retention time of
about 9.9 min (Fig. 3A). Most of the 4-nitrobenzyl
derivative of the reaction product was associated with authentic
4-nitrobenzyl ester of nonanoic acid (Fig. 3B), whereas
the only minor peak that was associated with authentic 4-nitrobenzyl
oxime of nonanal was detected (Fig. 3C). When these
samples were coinjected with authentic derivatives, an enhancement of
each peaks was observed. Amounts of produced nonanoic acid and nonanal
were estimated at 88.9 and 6.1 nmol, respectively. These results
suggest that the nonanol as substrate was oxidized to nonanoic acid
with nonanal as a possible intermediate by this reaction system.
Figure 3:
Identification of the reaction products of
alcohol:NADoxidoreductase by high performance liquid
chromatography. The UV-absorbing derivatives of nonanoic acid, nonanal,
and the reaction products were prepared as described in the text. A, the 4-nitrobenzyl derivatives of nonanoic acid (10 nmol)
and nonanal (30 nmol). B, 4-nitrobenzyl ester of the reaction
products. 10 µl of the sample (100 µl) was injected. C, 4-nitrobenzyl oxime of the reaction products. 50 µl of
the sample (100 µl) was injected.
Table 2shows kinetic constants of the peroxisomal
alcohol:NAD oxidoreductase for straight chain
aliphatic alcohols. The same preparation of the enzyme in Fig. 2was used. The K
values of the enzyme
toward straight chain aliphatic alcohols decreased with increasing
chain length. The enzyme has K
values in the
millimolar range only for ethanol and in the micromolar range for
longer chain length aliphatic alcohols
(C
-C
) (Table 2). On the other
hand, the enzyme showed high activities toward medium chain length
aliphatic alcohols (C
-C
), showing the
maximum activity on nonanol (C
). The enzyme showed low
activities toward longer chain length
(C
-C
) or shorter chain length
(C
-C
) alcohols (Table 2). Methanol
did not serve as the substrate even at the high concentration of 2 M. NADP did not serve as a cofactor in all experiments.
Table 3shows the effect of inhibitors (1 and 10 mM)
on alcohol:NAD oxidoreductase activity of the same
preparation in Table 2. The enzyme activity was strongly
inhibited by cyanide and N -Ethylmaleimide. However the
activity was not affected by addition of iodoacetate, pyrazole
(inhibitor of alcohol dehydrogenase)(19) , or sodium azide
(inhibitor of catalase) (20) to the incubation mixture (Table 3).
In the present study, alcohol:NAD oxidoreductase was found to be present in the peroxisomes of rat
liver. The enzyme was induced by the administration of a peroxisomal
proliferator. The enzyme was NAD
-dependent and
oxidized straight chain aliphatic alcohols of a variety of carbon chain
(C
-C
) and not inhibited by sodium azide,
an inhibitor of catalase (Table 3), showing that the enzyme
differs from the peroxisomal catalase. Catalase, in addition to the
decomposition of H
O
, can also catalyze the
oxidation of short chain alcohols to corresponding aldehydes by the
NAD
-independent peroxidatic activity, but cannot
oxidize higher primary alcohol(5) . Furthermore, peroxisomal
alcohol:NAD
oxidoreductase is also distinguishable
from cytosolic alcohol dehydrogenase and microsomal alcohol-oxidizing
systems as follows. 1) The peroxisomal enzyme is insensitive to
pyrazole (Table 3), a potent inhibitor for cytosolic alcohol
dehydrogenase(19) . 2) The microsomal ethanol-oxidizing system
catalyzes the NADP
-dependent oxidation(3) .
However the peroxisomal enzyme cannot utilize NADP
as
cofactor. 3) Recently, it has been reported that the alcohol
oxidoreductase that can metabolize long chain aliphatic alcohols
(C
-C
) to the corresponding fatty acids
with NAD
as cofactor is present in rat
liver(21) . The enzyme is located in the microsomal fraction
and named fatty alcohol:NAD
oxidoreductase. However,
the enzyme cannot oxidize ethanol(21) .
We do not know
whether the peroxisomal alcohol:NAD oxidoreductase is
involved in ethanol metabolism in vivo or not. Two forms of
alcohol dehydrogenase have been reported to be present in the cytosol
of rat liver, designed class I and class III
isozymes(22, 23) . Only class I isozyme is active with
ethanol at the blood concentrations of this
compound(22, 23) . Class I isozyme exhibits K
value of 1,400 µM for ethanol (22, 23) somewhat higher than that of the peroxisomal
enzyme. This minor difference suggests a potential role for the
peroxisomal activity in ethanol metabolism.
On the other hand, the
specific activity of the peroxisomal enzyme for octadecanol is nearly
identical with that of rat liver microsomal fatty
alcohol:NAD oxidoreductase (1.3 milliunits/mg
microsomal protein)(21) . Recently, it has been reported that
alkyl dihydroxyacetone phosphate synthase is mainly localized in
peroxisomes (24) . The enzyme catalyzes the biosynthesis of
ether lipids from acyl-dihydroxyacetone phosphate and fatty alcohol.
These reports and the present data suggest a potential role for the
peroxisomal activity in regulating the cellular levels of ether-linked
lipids. Quantitative data on the physiological role of the peroxisomal
alcohol:NAD
oxidoreductase are required.
Rizzo et al.(25) have reported that a fatty alcohol cycle
is present in human skin fibroblasts. In this cycle, fatty acyl-CoA is
converted to the corresponding alcohols by acyl-CoA reductase and
generated fatty alcohol is converted to the corresponding fatty acid by
fatty alcohol:NAD oxidoreductase.
Sjögren-Larsson syndrome, a metabolic disease
characterized by abnormal accumulation of fatty alcohols in skin
fibroblasts, congenital ichthyosis, and mental retardation, is caused
by a defect of fatty alcohol:NAD
oxidoreductase in
this cycle(26, 27) . Fatty alcohol:NAD
oxidoreductase activity has been reported to be associated with
the particulate fraction of skin fibroblasts homogenate(27) .
It is of interest whether this disease is caused by disorder of the
peroxisomes or not.