From the
The pathway for the peroxisomal
The
According to the
classical pathways of unsaturated fatty acid degradation, 7,10-16:2 and
3,6,9,12-18:4 would be formed, respectively, after one cycle of
9,12-18:2 and 5,8,11,14-20:4
During the
The results in
Fig. 1
show that the generation of acid-soluble radioactivity
formed during the
The above
data show that when polyunsaturated fatty acids, with their first
double bond at position 6, 7, or 9, are incubated with peroxisomes, in
the absence of NAD
It is not known in vivo how arachidonate distributes
itself between mitochondria and peroxisomes for
Studies, particularly
by Osmundsen and his colleagues
(6, 7) , have shown that
arachidonate is a relatively poor substrate for peroxisomal
Even though acids accumulated with their first double bond at
position 4, when arachidonate-derived lipoxygenase products or
arachidonate were incubated with peroxisomes, our results show that
peroxisomes are able to
Peroxisomal and mitochondrial pathways of
unsaturated fatty acid degradation are identical, but the enzymes are
all different. The original observation, that the removal of a double
bond at position 5 during mitochondrial fatty acid
When
we incubated peroxisomes with [1-
The metabolic
studies with 4,7,10-16:3 provides evidence that peroxisomes have
dienoyl-CoA isomerase, and, moreover, the data suggest that the enzyme
may be a different protein than is found in mitochondria. More
conclusive evidence for a peroxisomal dienoyl-CoA isomerase was
obtained by characterizing the products that accumulate when
incubations were carried out with and without NADPH. In this regard,
both 5,8-14:2 and 5,8,11,14-20:4 are metabolized via identical
pathways.
The following observations suggest that arachidonate is
degraded as shown in Fig. 8. 1) As expected, when peroxisomes
were incubated with 6,9,12-18:3 and 7,10,13,16-22:4, without
nucleotides, it was possible to isolate 3-hydroxy-6,9,12-18:3 and
3-hydroxy-7,10,13,16-22:4. 2) When arachidonate was incubated under
identical conditions, no polar metabolite was produced, but
2-trans-4,8,11,14-20:5 accumulated. This finding implies that
2,4-dienoyl-CoA reductase is required for the first cycle of
In summary, our
results suggest that both odd-numbered double bonds in arachidonate
require 2,4-dienoyl-CoA reductase and the dienoyl-CoA isomerase for
their removal during peroxisomal
-oxidation of arachidonic
acid (5,8,11,14-20:4) was elucidated by comparing its metabolism with
4,7,10-hexadecatrienoic acid (4,7,10-16:3) and 5,8-tetradecadienoic
acid (5,8-14:2) which are formed, respectively, after two and three
cycles of arachidonic acid degradation. When
[1-
C]4,7,10-16:3 was incubated with peroxisomes
in the presence of NAD
and NADPH, it resulted in a
time-dependent increase in the production of acid-soluble radioactivity
which was accompanied by the synthesis of
2-trans-4,7,10-hexadecatetraenoic acid and two
3,5,7,10-hexadecatetraenoic acid isomers. The formation of conjugated
trienoic acids suggests that peroxisomes contain
,
-dienoyl-CoA isomerase with the
ability to convert 2-trans-4,7,10-hexadecatetraenoic acid to
3,5,7,10-hexadecatetraenoic acid. When 1-
C-labeled
6,9,12-octadecatrienoic acid or 7,10,13,16-docosatetraenoic acid was
incubated without nucleotides, the 3-hydroxy metabolites accumulated,
since further degradation requires NAD
-dependent
3-hydroxyacyl-CoA dehydrogenase. When
[1-
C]5,8,11,14-20:4 was incubated under
identical conditions, no polar metabolite was detected, but
2-trans-4,8,11,14-eicosapentaenoic acid accumulated. When
NADPH was added to incubations, 3-hydroxy-8,11,14-eicosatrienoic,
2-trans-4,8,11,14-eicosapentaenoic,
2-trans-8,11,14-eicosatetraenoic, and 8,11,14-eicosatrienoic
acids were produced. Analogous compounds were formed from
[1-
C]5,8-14:2. Our results show that the removal
of double bonds from odd-numbered carbons in arachidonic acid thus
requires both NADPH-dependent 2,4-dienoyl-CoA reductase and
,
-dienoyl-CoA isomerase. One
complete cycle of 5,8-14:2 and 5,8,11,14-20:4
-oxidation yields,
respectively, 6-dodecenoic and 6,9,12-octadecatrienoic acids.
-oxidation of unsaturated fatty acids requires both
2,4-dienoyl-CoA reductase and
,
-enoyl-CoA isomerase, in addition to
the enzymes required for saturated fatty acid
degradation
(1, 2) . Peroxisomes contain a trifunctional
enzyme possessing
-enoyl-CoA hydratase,
3-hydroxyacyl-CoA dehydrogenase, and
,
-enoyl-CoA isomerase activities (3).
Peroxisomal 2,4-dienoyl-CoA reductase
(4) is a different protein
from the two mitochondrial isoforms of this enzyme
(5) . In their
comparative studies on peroxisomal
-oxidation of different
unsaturated fatty acids, Osmundsen and his colleagues
(6, 7) reported that the amount of acid-soluble radioactivity
produced from [1-
C]arachidonate was frequently
less than from other 1-
C-labeled acids. We also observed
that the rate of production of acid-soluble radioactivity from
[1-
C]5,8,11,14-20:4 was considerably less than
that from [1-
C]9,12-18:2 or
[1-
C]7,10,13,16-22:4
(8) . When
tritium-labeled arachidonic acid was incubated with peroxisomes,
Hiltunen et al.
(6) did not detect any 18-carbon
catabolite but a compound did accumulate that they suggested was either
5,8-14:2 or 4,7,10-16:3. We carried out similar studies and were able
to characterize both 4,7,10-16:3 and
2-trans-4,7,10-16:4
(8) . When
[U-
C]9,12-18:2 was incubated with peroxisomes,
both 7,10-16:2 and 5,8-14:2 accumulated
(8) .
-oxidation. However, Tserng and Jin
(9) reported that in mitochondria the double bond at position 5,
in a number of monounsaturated fatty acids, was directly removed via a
nucleotide-dependent 5-reductase. This pathway has now been modified to
show that it requires NADPH-dependent 2,4-dienoyl-CoA reductase, an
enzyme which previously was thought to be required for double bond
removal only at even-numbered carbons
(1) . In addition, a new
enzyme,
,
-dienoyl-CoA isomerase
is required (10, 11). This enzyme has recently been purified from
mitochondria by Schulz and co-workers
(12) , as well as by Tserng
and his collaborators (13). In the study reported here, we show that,
during the peroxisomal
-oxidation of arachidonate, the removal of
odd-numbered double bonds requires both NADPH-dependent 2,4-dienoyl-CoA
reductase and
,
-dienoyl-CoA
isomerase.
Materials
ATP, NAD, NADPH,
CoASH, Hepes, dithiothreitol, TES
(
)
, and
essentially fatty acid-free bovine serum albumin were from Sigma.
Lactate dehydrogenase and Nycodenz (Accudenz) were obtained,
respectively, from Boehringer Mannheim and Accurate Chemicals and
Scientific Corp. Arachidonic acid was from Nu-Chek Prep, while the
other unlabeled and 1-
C-labeled acids were made by total
synthesis
(14) . The methods described by Stoffel and Pruss
(15) were used to synthesize 3-hydroxy-8,11,14-20:3 and
2-trans-8,11,14-20:4. Authentic
2-trans-4-cis-10:2 was isolated from the seed oil of
the Chinese Tallow Tree
(16) .
Isolation of Peroxisomes and Mitochondria from Rat
Liver
Male Sprague-Dawley rats were maintained on a chow diet
containing 0.5% Clofibrate (prepared by Dyets, Inc., Bethlehem, PA) for
8 days prior to being killed. Peroxisomes were isolated in essence by
the method of Das et al.(17) , as described previously
(8). Briefly, liver was homogenized in 0.25 M sucrose, 0.1
mM EDTA, 0.1% ethanol, 10 mM TES, pH 7.5. The nuclei
and mitochondria obtained by centrifugation at 600 g for 10 min and 3,300
g for 10 min were discarded.
The light mitochondrial pellet that sedimented at 25,000
g for 15 min was washed with the homogenizing buffer and centrifuged
at 25,000
g for 12 min. One more washing was done
under the same conditions, except that the centrifugation time was
reduced to 10 min. The light mitochondrial fraction was resuspended in
the homogenizing medium in a volume corresponding to 1 ml/g liver. Two
ml of this suspension was layered on 15 ml of 35% Nycodenz (w/v),
containing 10 mM TES, 0.1% ethanol, and 0.1 mM EDTA,
pH 7.5, and centrifuged at 56,000
g for 50 min. The
peroxisomal pellet was suspended in the incubation medium which
contained 130 mM KCl, 20 mM Hepes, pH 7.2, and the
protein concentration was adjusted to 3 mg/ml. Protein was assayed with
the Coomassie Blue reagent (Pierce), using bovine serum albumin as a
standard. Purified mitochondria were isolated according to the
procedure of Johnson and Lardy
(18) , as modified by Broekemeier
et al.(19) . The purity of peroxisomes was determined
by marker enzyme analysis as described previously
(8) .
Mitochondrial contamination was less than 1% as determined by measuring
succinate cytochrome c-reductase in mitochondria and in the
Nycodenz-purified peroxisomes
(20) .
Peroxisomal Metabolism
To measure fatty acid
activation, peroxisomes (10 µg of protein) were incubated in a
total volume of 0.2 ml, at 37 °C for 2 min in a shaking water bath,
in a medium that contained 130 mM KCl, 20 mM Hepes
(pH 7.2), 10 mM MgATP, and 0.2 mM
CoASH. Reactions were initiated by addition of the sodium salt of the
fatty acid (2 Ci/mol) bound to bovine serum albumin in a 2:1 molar
ratio. Substrate concentration varied from 12.5 to 150 µM.
Reactions were terminated after 2 min by addition of 1 ml of
Dole's reagent (isopropyl alcohol/heptane/0.1 N
H
SO
, 40:10:1, v/v), followed by 0.35 ml of
water and 0.6 ml of heptane
(21) . The upper layer was discarded,
the bottom layer was washed three times with 1-ml aliquots of heptane,
by vortexing, and the bottom layer was transferred to scintillation
vials and counted in 10 ml of ACS II (Amersham). Maximum rates of
activation were calculated from Lineweaver-Burk plots. To measure
-oxidation, peroxisomes (300 µg of protein) were incubated at
37 °C in a shaking water bath in a medium that contained 130
mM KCl, 20 mM Hepes, 0.1 mM EGTA, 0.5
mM NAD
, 0.1 mM NADPH, 0.1
mM dithiothreitol, 0.4 mM CoASH, 10 mM
Mg
ATP, 20 mM pyruvate, and 2 units of
lactate dehydrogenase, pH 7.2
(8, 22) . Reactions were
initiated by addition of the sodium salt of the fatty acid (2 Ci/mol)
that was bound to bovine serum albumin in a 2:1 molar ratio. The final
concentration of fatty acid was always 100 µM. Aliquots of
200 µl were removed and added to an equal volume of 5%
HClO
. After 30 min at 4 °C, the samples were
centrifuged and 200 µl was counted to measure acid-soluble
radioactivity.
Metabolite Isolation and Characterization
Large
scale incubations were always carried out without NAD,
but with and without NADPH in order to isolate metabolites for
characterization. For each milliliter of incubation medium, 1.7 ml of
MeOH and 0.25 ml of 4 N NaOH was added. The contents were
stirred overnight, acidified with 0.25 ml of 6 N HCl, and 3.4
ml of CHCl
was added. The bottom layer was taken to dryness
under N
, and the free fatty acids were esterified by
stirring them overnight with 5% anhydrous HCl in MeOH (w/v). The methyl
esters were recovered by extraction with hexane. They were separated by
reverse phase HPLC by eluting a 0.46
25 cm Zorbax ODS column
with various concentrations of acetonitrile/water. The effluent (1
ml/min) was mixed with ScintiVerse LC (Fisher) (3 ml/min), and
metabolites were detected with a Beckman 171 radioisotope detector.
Appropriate fractions were collected from the column effluent, the
acetonitrile was removed under N
, and the metabolites were
recovered by extraction with 20% diethyl ether in hexane. Trace amounts
of water were removed by passing the combined extract through a Pasteur
pipette packed with granular Na
SO
. Ultraviolet
spectra were measured in MeOH using a Beckman DU-64 spectrophotometer.
The methyl esters of 3-hydroxy fatty acids were further derivatized by
reacting them with equal volumes of
N,O-bis(trimethylsilyl)trifluoroacetamide (Pierce
Chemical Co.) and pyridine for 30 min at 60 °C. Mass spectrometry
was carried out with a Hewlett-Packard Model 5970 mass selective
detector and a 5770 gas chromatograph containing a 30 m
0.25 mm
inside diameter, DB5-ms column (J and W Scientific). Injections were
made in isooctane in the splitless mode at 70 °C, and, after 3 min,
the oven temperature was programmed to increase, at 20 °C/min to
200 °C.
-oxidation of arachidonate, the synthesis of
the acyl-CoA derivatives of 5,8-14:2 and 4,7,10-16:3 occurs by a
CoASH-dependent thiolytic cleavage of precursor 3-ketoacyl-CoAs, rather
than by ATP-dependent activation. Rates of activation of 5,8-14:2,
4,7,10-16:3, and arachidonate were, respectively, 746, 557, and 283
nmol/min/mg of peroxisomal protein. Since activation rates were high,
it was assumed that acyl-CoA formation with all three substrates was
not rate-limiting when
-oxidation was assayed.
-oxidation of
[1-
C]5,8-14:2 was greater than for
[1-
C]5,8,11,14-20:4. These data suggest that the
chain length of the substrate plays a more important role in regulating
-oxidation than does a double bond at position 5. The generation
of acid-soluble radioactivity from
[1-
C]4,7,10-16:3 requires the participation of
NADPH-dependent 2,4-dienoyl-CoA reductase. As shown in Fig. 1,
there was a time dependent increase in the rate of
-oxidation when
incubations were carried out in the absence of NADPH. When NADPH was
added to the incubation, there was an increase in the generation of
acid-soluble radioactivity. Similar results were obtained when the rate
of
-oxidation of [1-
C]4,7,10,13,16,19-22:6
was compared with and without NADPH
(6) .
Figure 1:
The time-dependent -oxidation
[1-
C]5,8-14:2 (
),
[1-
C]5,8,11,14-20:4 (
),
[1-
C]4,7,10-16:3 without (
) and with
(
) NADPH. Peroxisomes (300 µg of protein/ml) were incubated
with 100 µM levels of fatty acids. At the indicated times,
200 µl was removed and added to 200 µl of cold 5%
HClO
. After centrifugation, 200 µl of the supernatant
was used to measure acid-soluble
radioactivity.
When
[1-C]4,7,10-16:3 was incubated with the
NAD
generating system and NADPH, the HPLC
radiochromatogram in Fig. 2A shows that three
radioactive metabolites eluted immediately prior to compound 4 which is
residual substrate. When [1-
C]4-10:1, a product
that would be produced after five cycles of arachidonate
-oxidation, was incubated under identical conditions, it was
possible to detect only one metabolite in addition to unreacted
substrate (Fig. 3). The metabolite was isolated and its
ultraviolet and mass spectra were identical with authentic methyl
2-trans-4-cis-10:2. When
[1-
C]4,7,10-16:3 was incubated without
nucleotides, there was an increase in the accumulation of metabolites
versus when the complete system was used (Fig. 2, A
versus B). These incubation conditions were then used to
accumulate sufficient amounts of metabolites for characterization. The
ultraviolet spectrum of the methyl ester of compound 3
(Fig. 4A) was identical with that of methyl
2-trans-4-cis-10:2 (Fig. 4B). The mass
spectrum of the methyl ester had a base peak of m/z 79, a
small molecular ion at m/z 262 (2%) with other small ions at
m/z 230 (M - 32; 1%) and m/z 231 (M - 31;
1%). Compound 3 is thus methyl 2-trans-4,7,10-16:4. The
ultraviolet spectrum of compound 2, as shown in Fig. 5, had three
absorbance maxima at 257, 267, and 277 nm showing that it contained
three conjugated double bonds, none of which were in conjugation with
the carbonyl carbon
(23) . Its mass spectrum differed from
compound 3 primarily in that the molecular ion at m/z 262 was
more intense; i.e. 32%, and the base peak was at m/z 91. The ultraviolet and the mass spectra of compound 1 were
similar with compound 2 (data not shown). The initial product formed
during the
-oxidation of 4,7,10-16:3 is
2-trans-4,7,10-16:4; i.e. compound 3. Isomerization
of the 2-trans-4-cis-double bonds, by reversing the
reaction catalyzed by
,
-dienoyl-CoA isomerase, would
yield 3,5,7,10-16:4. Two isomeric products with this general structure
were characterized, but as yet it has not been possible to determine
the configuration of the double bonds in compounds 1 and 2 in
Fig. 2
A and 2B.
Figure 2:
Radiochromatograms obtained when 100
µM [1-C]4,7,10-16:3 was incubated
with 300 µg of peroxisomal protein with (A) and without
(B) NAD
and NADPH. After 30 min, the
incubations were terminated, and methyl esters were made and separated
by HPLC by eluting the column at 1 ml/min with acetonitrile/water
(85:15, v/v).
Figure 3:
A radiochromatogram showing that the
metabolism of [1-C]4-10:1 resulted in the
production of a single radioactive catabolite (compound 1). Peroxisomes
were incubated with 100 µM
[1-
C]4-10:1 in the presence of NAD
and NADPH. After 30 min, the reaction was terminated, and methyl
esters were made and separated by HPLC by eluting the column at 1
ml/min with acetonitrile/water (70:30, v/v).
Figure 4:
A, the ultraviolet spectra of metabolite
3 from Fig. 2; B, authentic methyl
2-trans-4-cis-10:2; C, the major catabolite
that was produced when [1-C]5,8,11,14-20:4 was
incubated without NAD
and NADPH as shown in Fig.
6A; and D, the major radioactive compounds which
co-eluted as shown in Fig. 7A.
Figure 5:
The ultraviolet spectrum of compound 2,
Fig. 2.
The above studies suggested that peroxisomes
have ,
-dienoyl-CoA isomerase
activity. Experiments were then designed to determine if the double
bond at position 5 was removed via the classical pathway of
-oxidation or if
,
-dienoyl-CoA isomerase was
required. Diczfalusy et al.
(24) reported that
3-hydroxy-9,12-18:2 accumulated when 9,12-18:2 was incubated with
peroxisomes in the absence of NAD
. When we incubated
[1-
C]6,9,12-18:3 with peroxisomes, without
NAD
and NADPH, a polar metabolite accumulated. The
mass spectrum of the methyl ester-trimethylsilyl ether derivative had a
weak molecular ion at m/z 380 (1%) and a major ion at m/z 365 (M - 15; 30%) showing that it is 3-hydroxy-6,9,12-18:3.
In a similar way, the mass spectrum of the polar metabolite derived
from [1-
C]7,10,13,16-22:4 had a molecular ion at
m/z 434 (2%) and a major ion at m/z 419 (M -
15; 37%). This compound is thus 3-hydroxy-7,10,13,16-22:4.
, that the expected 3-hydroxy
metabolite accumulates. When [1-
C]5,8,11,14-20:4
was incubated under the above conditions, the HPLC radiochromatogram in
Fig. 6A shows that it was not possible to detect a polar
metabolite. Two small radioactive peaks eluted, respectively, at 22.7
and 24 min. The major metabolite, which eluted immediately prior to
unmetabolized arachidonate, had an ultraviolet spectrum
(Fig. 4C) identical with methyl
2-trans-4-cis-10:2 (Fig. 4B). The mass
spectrum of the methyl ester had a weak molecular ion at m/z 316, which is two mass units less than for methyl 5,8,11,14-20:4.
Compound 3 is thus 2-trans-4,8,11,14-20:5, which cannot be
produced from arachidonate via the classical pathway of
-oxidation
since one cycle of
-oxidation would yield 3,6,9,12-18:4, using
only the enzymes required for saturated fatty acid degradation. The
accumulation of 2-trans-4,8,11,14-20:5 further implies that
peroxisomes have
,
-dienoyl-CoA
isomerase activity. If the dienoyl-CoA isomerase-NADPH-dependent
reductase pathway is required to remove the double bond at position 5
from arachidonate, the synthesis of the 3-hydroxyacyl-CoA would require
NADPH. The HPLC radiochromatogram in Fig. 6B shows that
it was possible to detect seven radioactive metabolites when
[1-
C]5,8,11,14-20:4 was incubated with
peroxisomes in the presence of NADPH in addition to unmetabolized
substrate (compound 5). Under these conditions, it was not possible to
detect acid-soluble radioactivity, which would require a complete cycle
of
-oxidation. The mass spectrum of the methyl
ester-trimethylsilyl ether of compound 1 had a molecular ion at m/z 408 (6%) and a major ion at m/z 393 (M - 15; 47%).
Its spectrum was identical with that of the methyl ester-trimethylsilyl
derivative of synthetic 3-hydroxy-8,11,14-20:3. Compound 4 is methyl
2-trans-4,8,11,14-20:5. Compounds 7 and 8 were identified by
comparing their retention times on HPLC and their mass spectra with
authentic standards. They are, respectively, methyl
2-trans-8,11,14-20:4 and methyl 8,11,14-20:3.
Figure 6:
HPLC
radiochromatograms of the products obtained when 100 µM
[1-C]5,8,11,14-20:4 was incubated without
(A) and with (B) NADPH. Peroxisomes (300 µg of
protein) were incubated for 30 min, the reactions were terminated, and
the methyl esters were made and separated by HPLC by eluting the column
at 1 ml/min with acetonitrile/water (85:15,
v/v).
When
[1-C]5,8-14:2 was incubated without nucleotides,
two radioactive metabolites eluted at 13.7 and 14.4 min
(Fig. 7A). They were collected together and, when
analyzed by GC-MS, it was possible to detect two compounds, both of
which had molecular ions at m/z 236, which is two mass units
less than for the molecular ion of methyl 5,8-14:2. The major
radioactive compound had a retention time identical with methyl
[1-
C]5,8-14:2. When it was isolated and analyzed
by GC-MS, two compounds were separated, one of which was unmetabolized
substrate while the second compound had a molecular ion at m/z 236. The ultraviolet spectrum of this composite sample
(Fig. 4D) shows that it contains methyl
2-trans-4,8-14:3. Since the compounds eluting at 13.7 and 14.4
min also had molecular ions at m/z 236, they undoubtedly are
2-trans-5,8-14:3 and 3,5,8-14:3. It was not possible to obtain
satisfactory mass spectra of the analogous 5,8,11,14-20:4 metabolites,
i.e. compounds 2 and 3 (Fig. 6B); but, most
likely, they are methyl 2-trans-5,8,11,14-20:5 and
3,5,8,11,14-20:5.
Figure 7:
HPLC
radiochromatograms of the products produced when 100 µM
[1-C]5,8-14:2 was incubated without (A)
and with (B) NADPH. Peroxisomes (300 µg of protein) were
incubated for 30 min, the reactions were terminated, and methyl esters
were made and separated by HPLC by eluting the column at 1 ml/min with
acetonitrile/water (85:15, v/v).
When [1-C]5,8-14:2 was
incubated with peroxisomes in the presence of NADPH, the HPLC
radiochromatogram in Fig. 7B shows that it was also
possible to detect several radioactive metabolites. The mass spectrum
of the methyl ester-trimethylsilyl ether of compound 1 had a molecular
ion at m/z 328 (2%) and an ion at m/z 313 (M -
15; 27%) showing that it is 3-hydroxy-8-14:1. Compounds 2 and 3 are
2-trans-5,8-14:3 and 3,5,8-14:3. Again, compound 4 was a
mixture of unmetabolized methyl 5,8-14:2 and methyl
2-trans-4,8-14:3. Compound 5, which corresponds to the
uncharacterized arachidonate-derived metabolite (compound 6;
Fig. 6B) was also not identified. Compounds 6 and 7 had
molecular ions respectively at m/z 238 and 240. By analogy
with the arachidonate-derived metabolites, these undoubtedly are methyl
2-trans-8-14:2 and methyl 8-14:1.
-oxidation.
Several types of evidence suggest that peroxisomes play a major role in
this process. Studies using a variety of cells have shown that the
lipoxygenase products, 12-hydroxy-5,8,10,14-20:4 (12-HETE) and
15-hydroxy-5,8,11,13-20:4 (15-HETE), are
-oxidized
(25, 26, 27, 28) . With
both substrates, the major or the only catabolite that accumulated had
its first double bond at position 4, i.e. from 12-HETE it was
8-hydroxy-4,6,10-16:3 and from 15-HETE it was 11-hydroxy-4,7,9-16:3.
When liver peroxisomes were incubated with 12-HETE,
8-hydroxy-4,6,10-16:3 accumulated as the sole catabolite
(29) .
Neither skin fibroblasts from patients with Zellweger's disease
(30) nor Chinese hamster ovary cells with peroxisomal
deficiencies
-oxidized 12-HETE
(31) . When tritium-labeled
5,8,11,14-20:4 was incubated with liver peroxisomes, Hiltunen et
al.
(6) reported that a radioactive catabolite was detected
which they suggested was either 5,8-14:2 or 4,7,10-16:3. Using the same
incubation conditions, we were able to characterize both 4,7,10-16:3
and 2-trans-4,7,10-16:4
(8) . When tritium-labeled
arachidonic acid was incubated with fibroblasts from control patients,
it was possible to detect tritium-labeled 4,7,10-16:3 in the culture
medium. It was not possible to detect this catabolite when fibroblasts
from a Zellweger's patient were used
(32) . The above
studies document that peroxisomes have the capability of
-oxidizing arachidonate and the lipoxygenase products produced
from arachidonate. They also show that a product always accumulates
that has its first double bond at position 4.
-oxidation when compared with other long chain acids where the
first double bond is at different positions. We obtained similar
results
(8) . In the study reported here we show that 5,8-14:2, a
catabolite of arachidonate
-oxidation, is more rapidly
-oxidized than is arachidonate. The presence of a double bond at
position 5 does not per se determine whether an acid will be
rapidly
-oxidized by peroxisomes. In vivo, it is possible
that 5,8-14:2 may never accumulate within peroxisomes if a precursor,
such as 4,7,10-16:3, is converted to an acylcarnitine by peroxisomal
carnitine acyltransferase
(33) and transported out of
peroxisomes. The immediate precursor of 5,8-14:2 is 4,7,10-16:3, the
-oxidation of which requires NADPHdependent 2,4-dienoyl-CoA
reductase. When [1-
C]4,7,10-16:3 was incubated
with peroxisomes, there was a time-dependent increase in the production
of acid-soluble radioactivity that was enhanced by the addition of
NADPH. It is not known why peroxisomes generate acid-soluble
radioactivity from [1-
C]4,7,10,13,16,19-22:6
(6) or [1-
C]4,7,10-16:3 when they are
incubated in the absence of NADPH. The generation of acid-soluble
radioactivity from [1-
C]4,7,10-16:3 or
[1-
C]4,7,10,13,16,19-22:6
(6) , in the
absence of exogenous NADPH, could be explained if peroxisomes contained
NADPH, when isolated by Nycodenz centrifugation. This seems unlikely
since when [1-
C]5,8,11,14-20:4 was incubated
with NAD
and either NADP
(8) or NADPH, the rates of production of acid-soluble
radioactivity were similar. The results suggest that when peroxisomes
are incubated with NAD
and either NADP
or NADPH, that a transhydrogenase may operate to maintain a level
of NADPH sufficient for NADPH-dependent reactions as is required for
the first cycle of 4,7,10-16:3 and 5,8,11,14-20:4
-oxidation.
-oxidize 4,7,10-16:3. It was not possible
to assay [1-
C]4-10:1
-oxidation by
measuring acid-soluble radioactivity due to solubility of the substrate
in 5% perchloric acid. The characterization of
2-trans-4-cis-10:2 shows that 4-10:1 is a substrate
for fatty acyl-CoA oxidase. Our results show that peroxisomes have the
capacity to remove the double bonds at positions 5, 8, and 11 from
arachidonate, and, most likely, they are also able to remove the double
bond at position 14.
-oxidation
requires a 5-reductase, has been modified. Both Schulz and his
co-workers
(10) , as well as Tserng and
collaborators
(11) , have shown that NADPH-dependent
2,4-dienoyl-CoA-reductase and a new enzyme,
,
-dienoyl-CoA isomerase, are
required. Both groups of investigators have purified a mitochondrial
protein with this enzymatic activity
(12, 13) .
C]4,7,10-16:3,
it was possible to isolate two 16-carbon acids containing a conjugated
triene structure. The implication of this finding is that
2-trans-4,7,10-16:4 was isomerized to 3,5,7,10-16:4 via
reversal of the
,
-dienoyl-CoA
isomerase-catalyzed reaction. When 5-8:1, a product formed after five
cycles of
-oxidation of 9,12,15-18:3, was converted to 3,5-8:2, it
was a substrate for mitochondrial dienoyl-CoA isomerase but it was
inactive as a substrate with Nycodenz-purified peroxisomes
(12) .
The authors note that the results either indicate that peroxisomes do
not have dienoyl-CoA isomerase activity or they do not recognize a
short chain substrate. The same investigators reported that only small
amounts of 2-trans-4-trans-8:2 were converted to the
3,5-8:2 isomer by their purified dienoyl-CoA isomerase. It thus appears
that the synthesis of the 16:4 isomeric conjugated trienes could not be
formed by reversal of the dienoyl-CoA isomerase. There are two
important differences in the studies reported by Schulz and colleagues
(12) versus the studies reported here. The metabolism
of 2-trans-4,7,10-16:3 to 3,5,7,10-16:4 results in the
synthesis of a stable conjugated triene system. When either
[1-
C]4,7,10,13,16,19-22:6 or
[1-
C]4,7,10,13,16-22:5 were incubated with
peroxisomes, three radioactive metabolites also eluted immediately
prior to unmetabolized substrate.
(
)
When
[1-
C]4-10:1, a short chain acid, was incubated
with peroxisomes, it was only possible to isolate
2-trans-4-cis-10:2 suggesting that it was not
converted to the 3,5-10:2 isomer. It is not yet known whether the
mitochondrial dienoyl-CoA isomerase has the ability to metabolize
appropriate substrates into conjugated trienes.
-oxidation. 3) When incubations contained NADPH, a polar
catabolite accumulated, that was shown to be 3-hydroxy-8,11,14-20:3. 4)
When incubations contained NADPH, both 2-trans-8,11,14-20:4
and 8,11,14-20:3 were also produced. The 2-trans-8,11,14-20:4
serves as a substrate for the trifunctional enzyme with hydratase
activity, to yield 3-hydroxy-8,11,14-20:3. In addition, NADPH-dependent
reduction will yield 8,11,14-20:3. In 1974, Kunau and Bartnik
(34) showed that when mitochondria were incubated with a variety
of acids with their first double bond at position 4, that products were
produced containing the same number of carbon atoms, but the double
bond at position 4 was reduced. They subsequently partially purified a
protein with 2-trans-reductase activity that was different
from 2,4-dienoyl-CoA reductase
(35) . Our results suggest that
peroxisomes have this activity. Horie et al. (36) showed that
when peroxisomes were incubated with reduced pyridine nucleotides and
acetyl-CoA, they had an acetyl-CoA-dependent chain elongation system.
The conversion of 2-trans-8,11,14-20:3 to 8,11,14-20:3 is, in
essence, the last step in this process.
Figure 8:
The proposed pathway for the peroxisomal
-oxidation of arachidonic acid. The enzymes in this pathway are
fatty acid oxidase (1), the trifunctional enzyme with
,
-enoyl-CoA isomerase activity
(2),
,
-dienoyl-CoA
isomerase (3), NADPH-dependent 2,4-dienoyl-CoA reductase
(4), the trifunctional enzyme (5 and 6), and
an NADPH-dependent 2-trans-acyl-CoA reductase
(7).
It should be noted that one
complete cycle of arachidonate -oxidation in peroxisomes yields
6,9,12-18:3. This catabolite, as well as 8,11,14-20:3, produced via
NADPH-dependent reduction of 2-trans-8,11,14-20:4, are the two
immediate precursors for the synthesis of arachidonate in the
endoplasmic reticulum. We reported that when 7,10,13,16-22:4 was
incubated with microsomes and peroxisomes, it was rapidly
-oxidized to 5,8,11,14-20:4. However, once arachidonate was
produced, it was preferentially transported out of peroxisomes and used
as a substrate for microsomal acylation into
1-acyl-sn-glycero-3-phosphocholine rather than serving as a
substrate for continued
-oxidation
(8) . The possibility
thus exists, that when arachidonate is metabolized to 6,9,12-18:3 and
8,11,14-20:3, these acids may move out of peroxisomes for conversion
back to arachidonate in the endoplasmic reticulum.
-oxidation. It remains to be
determined, in vivo, how arachidonate partitions itself
between mitochondria and peroxisomes for
-oxidation. Additional
mitochondrial studies are also required to determine what fractional
amount of arachidonate is degraded via the classical pathway versus the newly described pathway that requires the two ancillary
enzymes
(9, 10, 11, 12, 13) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.