Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 1030-1036
FATTY Miguel
ACID
Bronfman, Department
OXIDATION Nibaldo
C. Inestrosa
of Cell
Biology,
de Chile, Received
April
BY HUMAN LIVER
Casilla
PEROXISOMES
and
Federico
Universidad
114-D,
Leighton
Catblica
Santiago,
Chile
30, 1979
SUMMARY A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acylCoA oxidase, its characteristic enzyme, acts maximally on C12-C18 saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already estab1ishe.d properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans. INTRODUCTION The has
not
acids
role been
also The
known
been
rats
also
they
In lism
from
rat fact, and
some drugs, eventually
and
proliferative
these
to evaluate their
of
this
is
have the
their presence
properties,
not
role
with
in must
acid
be studied.
1030
in the
liver
exert
in
so,
humans acid
since
detected
in
(6,7) in
Furthermore,
human
lipid
system,
liver human
(9).
composition
hypolipidemic
oxidizing
(3,5).
fatty
been
(8).
of peroxisomes
a fatty
rat
p-oxidation
enzymatic
to
(3,4)
also
not
Nafenopin
contribution
compounds
necessarily
has
a different
by Clo-
in
acid
fatty
inducible
peroxisomal
fatty
0006-291X/79/111030-07$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
the
of lipids
oxidize
is
drugs
response
treated
to understand
its
proliferation
mitochondrial
peroxisomes
which
(I)
peroxisome which
metabolism
peroxisomes
hypolipidemic
However
stimulate
the
other
effect
patients
liver
system
(2)
(1).
in
In rats,
to an enhancement
and a peroxisomal biopsies
humans.
to induce
attributed system
peroxisomes
insensitive
hypolipidemic
oxidizing
and
in
Nafenopin
(I),
drugs
by liver
studied
by a cyanide
fibrate
has
played
metaboaction and
of
Vol. 88, No. 3, 1979
The zes
BIOCHEMICAL
peroxisomal
fatty
acid
actions
which,
sembles
that
acyl-CoA
fatty
p-oxidation with
mediated to the
peroxisomes
by a fatty
fatty
(2,121.
acids
protein
of
system has
in
been
146,000
the
a diet
The present peroxisomal of its MATERIAL
fatty
report acid
characteristic
(IO).
The
in
mitochondria
oxidase,
oxidase,
control
in
saturated
establishes oxidizing enzyme
desaturation
of
is
(II),
mainly
rate
and
of rats
acids
the
in
on C,2-C18 as a flavoenzyme
in
which (2)
the
of
the
system
or 2.4
fold
liver
of
(14).
existence
acyl-CoA
re-
catalyzed
limiting
administration
system , and fatty
of re-
reaction,
characterized
fatty
cataly-
by a dehydrogenase
active
the
liver
sequence
LX,/? -
chain
(2)
by Nafenopin
rat
oxidative
as the
rats
from
first
partially
appears
RESEARCH COMMUNICATIONS
insensitive
the
acyl-CoA
T-fold rich
of
transport
mw (131, of
system
electron
This
liver
enhanced
by feeding
exception
of mitochondria
coupled
oxidizing
by a cyanide
the
derivative,
tightly
acid
AND BIOPHYSICAL
in
describes
human
a
some properties
oxidase.
AND METHODS
Liver biopsies were obtained from four patients with normal liver function tests undergoing surgery for uncomplicated gall-stone or gastroduodenal ulcer disease. Informed consent was obtained following the procedures established by the Ethics Committee from the Medical School of the Universidad Catblica de Chile. The biopsies, obtained at the beginning of surgery, were placed in ice cold 0.25 M-sucrose. Homogenization was performed in 0.25 M-sucrose containing 3 mM-imidazole HCl, pH 7.4, and 1 mM-dithiothreitol at 20-2596 w/v tissue concentration. The homogenates were fractionated in continuous Metrizamide density gradients by a modification of the procedure developed by Wattiaux et al. for the isolation of lysosomes from an enriched subcellular fraction (15). 0.5 ml aliquots of the homogenate were layered on top of gradients extending from 1.10 to 1.28 g/ml in tubes from an SW-65 rotor (Beckman Instruments Inc.) and were centrifuged 90 min at 39,000 r-pm, 4OC. The activity of the peroxisomal fatty acid oxidizing system was determined from palmitoyl-CoA dependent NAD+ reduction, as described by Lazarow & de Duve (3) with some modifications (2). The assay medium contained, at pH 8.3, 60 mM-Tris HCl, 24 pM-palmitoyl-CoA, 120 ,uM-FAD, 370 PM-NAD+, 94 mM-nicotinamide, 2.8 mM-dithio50 PM-CoA, threitol, 2 mM-potassium cyanide, and 0.15 mg/ml of bovine serum albumin in a final volume of 0.8 ml. The enzyme containing samples were diluted to 1/2 with 1% (w/v) Triton X-100 and 5-20 ~1 was added to each test. Units correspond to pmol NAD+ reduced . min-1. g wet wt. liver-l. The activity of the fatty acyl-CoA oxidase was determined polarographically from palmitoyl-CoA dependent oxygen consumption. The assay medium contained, at pH 8.3, 100 mM-Tris HCl,
1031
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 mM-potassium cyanide, 200 PM-NAD+, 31 PM-palmitoyl-CoA, 33 mM-nicotinamide, 170 PM-CoA, 50 PM-FAD, 0.6 mg/ml of bovine serum albumin, and 0.01% (w/v) Triton X-100 in a total volume of 1.6 ml. After addition of IO-50 ~"1 of the enzyme containing sample, the basal 02 uptake was recorder for 5 min, and the reaction started by addition of palmitoyl-CoA (2). Units correspond to pmol 02 consumed . min-1. g wet wt. liver-l. Established procedures were employed for the determination of marker enzymes: catalase (16) for peroxisomes , glutamate dehydrogenase (16) for mitochondria, NADPH cytochrome c reductase (17) for endoplasmic reticulum microsomes, and acid pFosphatase (18) for lysosomes. Protein was measured as described by Wattiaux et al. (15) using bovine serum albumin as standard. Crude fatty acyl-CoA oxidase preparations were made diluting the homogenates with 4 volumes of 100 mM-Tris HCl pH 8.3 and rehomogenizing in a tissue desintegrator (Virtis 45, The Virtis Co., Gardiner, New York). The supernatant obtained after centrifugation, 30 min at 100,000 g, was made 50% w/v with (NH412 SO4. The precipitate, collected by centrifugation, was dissolved and dialyzed overnight in the same buffer. This preparation frees the enzyme of some components responsible for high blank readings in the assay. The final specific activity was twice that of the homogenate. Metrizamide (2-(3-acetamido-5-N-methylacetamido-2-4-6 triiodobenzamido)-2-deoxy-D-glucose) was obtained from Nyegaard and CO., Oslo. Substrates and coenzymes were from Sigma Chemical Co., St. Louis, Missouri. RESULTS In
AND DISCUSSION four
oxidase,
liver
with liver;
system,
measured
2 0.06
the
+ 0.05 The
activity
(S.D.)
(S-D.)
the
liver,
palmitoyl-CoA
I).
The
gave
good
resolution
of peroxisomes
microsomes
and
reticulum activity system, top
of fatty follows
fractions
subcellular
together
rat
were
0.81
2 0.07
the
bound with
the
catalase. rest
of
1032
oxidizing was
liver,
applying
(S.D.)
in
was clearly
developed
mitochondria,
endoplasmic one half
of
the
fatty
acid
oxidizing
The
other
half,
stays
catalase
and
the
liver
peroxisomal
procedure
of
the
and
detected
Approximately and
acid
activities
from
oxidase
(S.D.)
(2).
fractionation
lysosomes.
acyl-CoA particle
normal
as substrate,
(Fig
acyl-CoA
NAD+ reduction,
In
of
fatty
+ 0.06
fatty
respectively
distribution
novel
of
was 0.16
dependent
found
units/g
using
activity
peroxisomal
liver.
values
subcellular
homogenates,
of
units/g the
the
as substrate,
as palmitoyl-CoA
same procedures, 0.81
studied,
palmitoyl-Cob
units/g
0.24
biopsies
of
the
the
in
the
other
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20
10 DPH
cyt. c 20
10
20
,lO
-20
-10 110 FIGURE
115
1.20 1.25 110 115 Density (g/ml)
1.20
1.25
1
Subcellular distribution of fatty acyl-CoA oxidase (FAO) and of the peroxisomal fatty acid oxidizing system (PFAOS). Complete homogenate, from human liver,fractionated in a continuous density gradient of Metrizamide. The distribution of peroxisomes,mitochondria, endoplasmic reticulum microsomes and lysosomes was determined from marker enzymes, catalase, glutamate dehydrogenase, NADPH cytochrome c reductase and acid phosphatase, respectively. Ordinates represent average frequency of the components for each fraction, Q/EQAp where Q represents the activity found in the fraction, CQ the total activity recovered and Ap the increment in density from top to bottom of the Frequency is plotted against gradient in the centrifuge tube (20). density in a histogram form. The hatched area represents the proportion of the total activity of each component recovered in the top 0.5 ml of the tube contents which correspond to the volume of the initial homogenate layer.
marker
enzymes.
better
resolution
fractions, lase, soluble fraction attributed
it fatty
In
separate
of
organelles
was established acyl-CoA
components of
the
Metrizamide
of
peroxisomal to
enzyme
the
and
release
the the
low
In
from
has
also
the
1033
in
density
oxidizing
homogenate.
enzymes
designed
equilibrating
that
oxidase,
gradients,
rat been
organelles
the
low
fraction system,
liver,
during
density of
cata-
follows
a large
observed
for
the soluble
and has
been
fractionation
Vol. 88, No. 3, 1979
Whatever
(5,191. release
might
oxidase. acid
BIOCHEMICAL
the
be,
It
Human fatty preparation
section.
This in
together
with is
little
our
liver
more
fatty
was
at
observation
of
acyl-CoA
in
the
acyl-CoA and
the
FAD (Fig
2).
that bound,
the
mitochondria. employing and
Methods
and a 5.5-fold The
effect
oxidase
suggests
similar
fatty
subcellular
Materials
kinetics
are
enzymes
characterized
assay
FAD weakly oxidases
of
all
in
establishing
with
fatty oxidase
further
linear
addition
than
pattern
as described gave
peroxisomal
acyl-CoA
no activity
oxidase
upon
fatty
that
of FAD, from
rat
human
flavoproteins
(13).
I
25
02
or
obtained
activity
for
a peroxisomal
preparation
I
FIGURE
have
a flavoprotein
and rat
that
acyl-CoA
a crude
responsible catalase
concluded
with
increase
affects
system
distribution,
liver
it
was
oxidizing
mechanism
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
50
75
i
16
16
Chain Lengthof substrate
Enzyme ( pl) 2
Effect of 50 PM-FAD on the rate of palmitoyl-CoA dependent oxygen consumption by a crude preparation of peroxisomal fatty acyl-CoA oxidase from human liver. The assays were performed as described in the Materials and Methods section, omitting NAT)+ and nicotinamide. FIGURE
3
Substrate specificity of human liver oxidase with saturated fatty acids, The rate of acyl-CoA dependent oxygen consumption was measured as described in the Materials and Methods section, omitting NAD+ and nicotinamide. A crude preparation of the enzyme was employed. Bars represent mean + S.D. of three independent determinations expressed as percent of the activity observed with palmitoyl-CoA.
1034
Vol. 88, No. 3, 1979
The with
substrate
three
fatty
described
for enzyme
90-100% in
of
rat
liver
crude
gave,
in
with
it
liver
has
fatty
system,
in
could
humans
by changes
with
(2).
The
the
that
is
the
present
work
oxidase
and
in
activities
findings
the
remains total that
the
human
saturated,
as well
mitochondria
to be established. of peroxiso-
of mitochondria
establishes of
that
that
of
were
contrast,
contribution
the
that
of peroxisomes
in
fatty lipid
and
(21).
presence
peroxisomal
possibility
capacity
In
of
the
that
suggest
pharmacologically
the
oleoyl-CoA
contribution
to
as already
to 40% of
metabolism
similar
supports
be controlled
induced
specific
amounts
the
oxidation
and
With
relative
for
31,
(2,12).
metabolism
suggested
(Fig
palmitoyl-CoA.
These in
acid
acyl-CoA
oxidizing
enzyme
was checked
activities
C12-C18
oleoyl-CoA
involved
been
conclusion,
of
with
oxidase
Maximal with
observed
fatty
acyl-CoA
preparation,
acids.
to
mes to palmitate In
each
are fatty
fatty
liver
palmitoyl-CoA
peroxisomes rats,
rat
activity
peroxisomes
and
of
observed
activities the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
preparations.
were
the
as unsaturated
In
crude
acids
the
liver
observed
specificity
different
saturated
human
BIOCHEMICAL
human
acid metabolism
physiologically
to oxidize
fatty
acids. ACKNOWLEDGEMENTS This work was supported by the Fondo de Investigaciones de la Universidad Cat6lica de Chile, proyecto 212/77, and by funds from the American States Organization. We thank Drs. F. Nervi for the clinical monitoring of the human volunteers, and R. Wattiaux for Metrizamide and a pre-publication copy of his work. The skilful participation of Cecilia Necochea and Maria Nelly Morales is gratefully acknowledged. REFERENCES I.
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