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(Received for publication, September 6, 1995; and in revised form, December 4, 1995) From the
The subcellular localization of L-lactate dehydrogenase
(LDH) in rat hepatocytes has been studied by analytical subcellular
fractionation combined with the immunodetection of LDH in isolated
subcellular fractions and liver sections by immunoblotting and
immunoelectron microscopy. The results clearly demonstrate the presence
of LDH in the matrix of peroxisomes in addition to the cytosol. Both
cytosolic and peroxisomal LDH subunits have the same molecular mass
(35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic
LDH antibody. As revealed by activity staining or immunoblotting after
isoelectric focussing, both intracellular compartments contain the same
liver-specific LDH-isoforms (LDH-A Lactate dehydrogenase (L-lactate:NAD The association of LDH
with rat liver peroxisomes (PO) was first suggested by the group of
Tolbert and co-workers(10) . However, the methodology used by
those authors did not allow an unequivocal conclusion, so that in
recent years it has generally been assumed that the LDH activity in
peroxisomal fractions is due to the adsorption of the cytosolic enzyme
to the outer surface of the peroxisomal membrane (11) . Whereas
PO in rat liver contain several dehydrogenases utilizing NAD In the present study, we have raised a monospecific
antibody against cytosolic rat liver LDH and demonstrate the presence
of the enzyme in the matrix of rat liver PO by a combination of
biochemical and ultrastructural immunocytochemical techniques.
Additionally, the involvement of peroxisomal LDH in the reoxidation of
NADH produced by the
Enzyme activities were determined according to standard procedures:
catalase and
To unravel the alterations of the individual LDH-isoforms, in a
separate set of experiments, freshly isolated PO as well as cytosolic
fractions exhibiting the same LDH activities (10 mU) were treated with
proteinase K (3.3 mg/ml stock solution in metrizamide 1.23
g/cm
Figure 1:
Distribution of marker enzyme
activities (catalase, glycolate oxidase (
Figure 2:
Distribution of marker enzyme activities
(catalase, palmitoyl-CoA oxidase (AOx), LDH, and glycolate
oxidase (
Figure 3:
Distribution of LDH in subfractions of
highly purified peroxisomes obtained by Metrizamide density gradient
centrifugation. Lanes 1 and 2, untreated total
peroxisomes. Lane 1, polypeptide pattern after SDS-PAGE and
silver staining (5 µg protein); lane 2, immunoblot using
anti-LDH antibody (2 µg of protein). Lanes 3-5,
integral membrane protein fraction. Lane 3, polypeptide
pattern after SDS-PAGE and silver staining (4.8 µg of protein); lane 4, immunoblot using anti-PMP 22 antibody (2 µg of
protein); lane 5, immunoblot using anti-LDH antibody (2 µg
of protein). Lanes 6-8, matrix fraction. Lane
6, polypeptide pattern after SDS-PAGE and silver staining (5
µg of protein); lane 7, immunoblot using anti-catalase
antibody (0.5 µg of protein); lane 8, immunoblot using
anti-LDH antibody (2 µg of protein). Lanes 9-11,
core fraction. Lane 9, polypeptide pattern after SDS-PAGE and
silver staining (1 µg of protein); lane 10, immunoblot
using anti-urate oxidase antibody (0.5 µg protein); lane
11, immunoblot using anti-LDH antibody (2 µg of protein).
Molecular mass standards in kDa are as follows: 66, bovine
serum albumin; 45, ovalbumin; 24, trypsinogen; 18.5,
Figure 4:
A, Western blot showing the presence of
LDH polypeptides with identical molecular masses in PO and the cytosol.
The following protein amounts were loaded per lane: cytosol (C), 0.25 µg; highly purified peroxisomes (P), 2
µg. B, isoelectric focussing gel stained for LDH activity
with the NBT-method, depicting LDH-A
Figure 5:
Influence of increasing concentrations of
KCl on the extraction of LDH, catalase, and protein from highly
purified PO. Freshly isolated PO (0.668 mg/ml) were diluted 10-fold in
5 mM MOPS, pH 7.4, 1 mM EDTA, 0.05% deoxycholate
containing different concentrations of KCl (20, 100, and 500
mM). After incubation for 15 min on ice, the mixtures were
centrifuged for 60 min at 100,000
Figure 6:
Influence of different physical
pretreatment conditions on the accessibility of LDH and catalase in PO
as revealed by proteolysis.
Substantial evidence for the
intraperoxisomal localization of LDH was provided by the differential
kinetics of degradation of the cytosolic and peroxisomal enzyme as
revealed by IEF (Fig. 7). Whereas the cytosolic LDH activity was
completely abolished after 15 min of protease treatment, the
peroxisomal LDH was only slightly affected even after 60 min (Fig. 7A). Only after lysis of PO with 1% Triton X-100
did the particle-bound LDH also disappear with similar kinetics as its
cytosolic counterpart (Fig. 7B).
Figure 7:
A,
kinetics of proteolysis on cytosolic and peroxisomal LDH as revealed by
IEF. Freshly isolated undiluted PO and cytosolic fractions exhibiting
the same activities (10 milliunits) were treated for different time
intervals with 0.33 mg/ml proteinase K, followed by IEF and activity
staining. Protein amounts corresponding to 5 milliunits of LDH activity
were loaded per lane: cytosol (C), 1.5 µg; highly purified
peroxisomes (P), 12 µg. The incubation times are indicated
by the numbers given in subscript. B, kinetics of proteolysis
of peroxisomal LDH (P) after lysis of the cell organelles by
1% Triton X-100. The incubation times are given by the numbers in
subscript.
Figure 8:
a
and b, electron micrographs of rat liver sections incubated
with the anti-LDH antibody followed by protein A-gold. Note the
localization of gold particles in the peroxisomal matrix (PO)
and the cytoplasm. Mitochondria (MITO) and lysosomes (LYS) are not labeled. c, control section labeled
with the antibody to catalase revealing exclusive peroxisomal
localization of catalase. d, control section incubated with
the appropriate LDH-preimmune serum.
The presence of LDH protein in isolated
peroxisomal fractions is demonstrated in Fig. 9. The
heterogeneity of LDH labeling in the peroxisomal fraction is clearly
visible in Fig. 9, a and b. Cores and
contaminating mitochondria are not labeled (Fig. 9b).
As revealed by higher magnification, only few gold particles are
attached to the cytosolic surface of the peroxisomal membrane (Fig. 9c), suggesting that the bulk of LDH in the
isolated fractions is intraperoxisomal. In quantitative counts of gold
particles, approximately 20% of all gold particles were associated with
either side of the peroxisomal membrane, thus confirming that at least
80% of labeling was truly intraperoxisomal. The appropriate controls
with anti-catalase and anti-LDH preimmune serum are shown in Fig. 9, d and e.
Figure 9:
a and b, highly purified PO
incubated with the anti-LDH antibody followed by protein A-gold and
silver intensification. Note the heterogeneous labeling of isolated
peroxisomes (PO). Two mitochondria (asterisk), and an
isolated core (arrowheads) are unlabeled. c, higher
magnification view of isolated peroxisomes labeled for LDH with 6-nm
gold particles. Note the presence of a few gold particles attached to
the cytoplasmic surface of the peroxisomal membrane (arrowheads) in addition to the labeling of the matrix. d and e, purified PO incubated with an antibody to catalase
and LDH-preimmune serum.
Figure 10:
Western blot of highly purified
peroxisomal fractions (2 µg of protein) from control rats (Co) and rats treated for 14 days with 75 mg/kg/d bezafibrate (Bz) incubated with the anti-LDH antibody. Molecular mass
standards in kDa are as follows: 45, ovalbumin; 24,
trypsinogen; 18.5,
In Table 3-V the influence of PO-associated LDH on the
reoxidation of NADH produced by peroxisomal
In the present study, the intracellular distribution of L-lactate dehydrogenase in rat hepatocyes was studied by three
different approaches: (a) analytical subcellular fractionation
with determination of enzyme activity, (b) immunodetection of
LDH in isolated subcellular fractions using a monospecific antibody, (c) immunoelectron microscopy applied to liver sections and to
isolated peroxisomal fractions. The results clearly demonstrate that
LDH is present in the matrix of rat liver peroxisomes in addition to
the cytosol. Moreover, the data presented in this study suggest
strongly that the peroxisomal LDH is directly coupled to the
reoxidation of the NADH generated by the palmitoyl-CoA
An association of LDH with PO was first reported by
the group of Tolbert and co-workers(10) . They stated, however,
that the possibility of LDH being associated to the surface of PO could
not be ruled out by the methods used in their studies. They found only
0.6% of the total LDH activity in intact PO, which after correction for
particle breakage during subcellular fractionation could make up as
much as 1.5% of the total activity. Since the peroxisomal isoform
(LDH-A In our earlier studies(18) , we
consistently found 1.5-2% of the total LDH activity in the crude
peroxisomal fraction. The latter is separated into two peaks after
density gradient centrifugation in an exponential metrizamide
gradient(23) . The first peak of LDH colocalized with the major
peroxisomal peak (fractions 2 and 3;
In a series of separate
experiments, a direct involvement of peroxisomal LDH in the reoxidation
of NADH produced by the If indeed the peroxisomal LDH plays a role
in regulating the peroxisomal NAD
Figure 11:
Proposed shuttle mechanism for the
transfer of reducing equivalents between PO and cytoplasm. 1-3, intermediates of the
Several LDH gene-related sequences, which may have arisen by gene
duplication of the original functional LDH gene have been reported for
the LDH-A and LDH-B genes(2, 46) . Until now, however,
only a part of these LDH gene-related sequences have been cloned,
sequenced, and characterized as nonfunctional, processed
pseudogenes(3, 47) . Therefore, in spite of
similarities in respect to kinetics, electrophoretic mobility and
antigenicity between the peroxisomal and cytosolic LDH's, the
possibility for the existence of functionally active peroxisomal LDH
genes should not be overlooked. For members of the closely related
malate dehydrogenase family, a separate gene has been found for each
isozyme localized in a different cell compartment (44) . Two
distinct targeting signals for peroxisomal matrix proteins have been
identified so far: a C-terminal tripeptide (SKL-variant; PTS1) and an
N-terminal PTS2(48) . Furthermore, the peroxisomal proteins do
not seem to require unfolding prior to import and can even be
translocated over the peroxisomal membrane as
oligomers(49, 50, 51, 52) . In
addition, epitope-tagged truncated subunits of peroxisomal thiolase,
lacking the PTS2 targeting signal, could be imported into yeast
peroxisomes in association with normal subunits containing the
N-terminal targeting signal (piggyback import)(51) . Similar
results were described for trimeric chloramphenicol
acetyltransferase-PTS1 (±) chimeras(52) . Thus, one
could speculate that a targeting signal in LDH isoform A would be
sufficient to direct both A The cloning and complete sequencing of the cDNA
for peroxisomal LDH-A (and -B) may resolve this question and may
clarify which type of targeting signal (PTS1 or PTS2) is conducting the
specific LDH-isoforms to the peroxisomes.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3846-3855
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
L
-Lactate
Dehydrogenase A- and A
B Isoforms Are Bona Fide
Peroxisomal Enzymes in Rat Liver
EVIDENCE FOR INVOLVEMENT IN INTRAPEROXISOMAL NADH REOXIDATION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
> LDH-A
B)
with the peroxisomes comprising relatively more LDH-AB than
the cytosol. Selective KCl extraction as well as resistance to
proteinase K and immunoelectron microscopy revealed that at least 80%
of the LDH activity measured in highly purified peroxisomal fractions
is due to LDH as a bona fide peroxisomal matrix enzyme. In combination
with the data of cell fractionation, this implies that at least 0.5% of
the total LDH activity in hepatocytes is present in peroxisomes. Since
no other enzymes of the glycolytic pathway (such as phosphoglucomutase,
phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase)
were found in highly purified peroxisomal fractions, it does not seem
that LDH in peroxisomes participates in glycolysis. Instead, the marked
elevation of LDH in peroxisomes of rats treated with the hypolipidemic
drug bezafibrate, concomitantly to the induction of the peroxisomal
-oxidation enzymes, strongly suggests that intraperoxisomal LDH
may be involved in the reoxidation of NADH generated by the
-oxidation pathway. The interaction of LDH and the peroxisomal
palmitoyl-CoA -oxidation system could be verified in a modified
-oxidation assay by adding increasing amounts of pyruvate to the
standard assay mixture and recording the change of NADH production
rates. A dose-dependent decrease of NADH produced was simulated with
the lowest NADH value found at maximal LDH activity. The addition of
oxamic acid, a specific inhibitor of LDH, to the system or inhibition
of LDH by high pyruvate levels (up to 20 mM) restored the NADH
values to control levels. A direct effect of pyruvate on palmitoyl-CoA
oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes
individually in separate assays. An LDH-based shuttle across the
peroxisomal membrane should provide an efficient system to regulate
intraperoxisomal NAD/NADH levels and maintain the flux
of fatty acids through the peroxisomal
-oxidation spiral.
oxidoreductase (LDH); (
)EC 1.1.1.27) is a tetrameric
protein catalyzing the reversible conversion of pyruvate to lactate.
The enzyme uses NAD/NADH as cofactor and exists in six
isoforms, five depending on the combination of the two subunits A
(muscle) and B (heart), each with a molecular mass of 35 kDa (1) and an additional homotetrameric LDH-C
(testis)-isoform(2, 3) . The three different
subunits (A, B, and C) are encoded by three structural genes, which
most probably originated from a common ancestral gene during evolution.
The expression of the LDH genes is developmentally regulated and
tissue-specific(4) . The liver-specific isoforms are
LDH-A and LDH-AB, which are mainly localized to
the cytoplasm of hepatocytes. There are several reports, however, on
the localization of LDH in other cell compartments. Whereas it is now
generally accepted that the tyrosine-phosphorylated LDH-A is localized to the nucleus and functions as a single-stranded
DNA-binding protein(5, 6) , the debate on the presence
of LDH as a bona fide enzyme in other cell organelles has not been
settled(7, 8, 9) . as cofactor such as (a) 3-hydroxyacyl-CoA
dehydrogenases(12) , (b)
-glycerol phosphate
dehydrogenase(13) , and (c) alcohol
dehydrogenase(14) , a peroxisomal enzyme system for the
reoxidation of NADH has not been described. Since the peroxisomal
membrane seems to be permeable to NADin
vitro(15) , it has been suggested that NADH, generated in
PO is reoxidized in the cytosol after passage across the peroxisomal
membrane(11) . Most recently, however, van Roermund et
al. (16) have shown that in vivo the peroxisomal
membrane in the yeast Saccharomyces cerevisiae is impermeable
to NAD
/NADH(16) . Moreover, Osmundsen et
al. (17) noted that the addition of pyruvate to an in
vitro peroxisomal
-oxidation assay stimulates the
-oxidation of palmitoyl-CoA while addition of exogenous LDH had no
effect. Since the exact mechanism of stimulation of -oxidation by
pyruvate remained ambiguous, we speculated that it could be due to the
presence of LDH inside the PO, since in our earlier studies this enzyme
was consistently found in the highly purified (98%) peroxisomal
fractions isolated by metrizamide density gradient centrifugation (18) .-oxidation system of this organelle is
demonstrated.
Materials
Blue dextran A coupled to agarose for
the isolation of LDH protein was obtained from Amicon (Witten,
Germany). Metrizamide for density gradients and nitrocellulose
membranes were purchased from Nycomed (Oslo, Norway) and Schleicher and
Schüll (Datteln, Germany), respectively. Proteinase
K and protein A were from Boehringer Mannheim, and BSA fraction V was
from Serva (Heidelberg, FRG). Constituents of enzyme assays (p-nitro blue tetrazolium, NAD, -ketoacids, glycolate,
palmitoyl-CoA, fructose 6-phosphate, glucose-6-phosphate dehydrogenase,
glucose 1-phosphate, glycerate 3-phosphate, phosphoglycerate kinase,
LDH-test Kit) were bought from Sigma and Boehringer Mannheim.
Titaniumoxisulfate was provided by Riedel-de-Haen (Seelze, Germany),
and glutaraldehyde was from Serva. All other chemicals used were
obtained from Merck (Darmstadt, Germany) and were of the purest grade
available.Animals and Drug Treatment
Normal Sprague-Dawley
rats of both sexes weighing 250 g, kept under normal laboratory
conditions, were used for all experiments. For the induction of the
peroxisomal
-oxidation enzymes, some rats were treated for 7 and
14 days with 75 mg/kg of bezafibrate (obtained through the courtesy of
Boehringer Mannheim), a dosage shown previously to be highly
effective(19) . All rats were fasted for 16 h prior to
sacrifice.Isolation of LDH and Raising of the Antibody
LDH
was isolated from total rat liver homogenates of untreated control
Sprague-Dawley rats according to the protocol described by Thompson et al.(20) . The purity of the enzyme was assessed by
SDS-PAGE. An antibody against purified LDH was raised in
rabbits(21) , and its specificity was confirmed by Western
blotting(22) .Cell Fractionation and Isolation of Highly Purified
Peroxisomes
Subcellular fractions comprising the heavy
mitochondrial (M), light mitochondrial or crude peroxisomal (L),
microsomal (P), and cytosolic (S) fractions were prepared by
differential centrifugation as described by Völkl
and Fahimi (18) . Briefly, rat livers were homogenized for 2
min at 1000 rpm in isotonic homogenization buffer (5 ml/g of tissue), 5
mM MOPS, 250 mM sucrose, 1 mM EDTA, 0.1%
ethanol, and protease inhibitors (0.2 mM phenylmethylsulfonyl
fluoride, 0.2 mM dithiothreitol, and 1 mM -aminocaproic acid) using an ice-cooled Potter-Elvehjem
homogenizer. In order to obtain highly purified PO, the L-fraction was subjected to density gradient centrifugation on
a continuous Metrizamide gradient (
= 1.10-1.26
g/cm
) as modified by Lüers et
al.(23) , spun in a vertical rotor (VTi 50, Beckman
Instruments) at an integrated force of 1.252 
10
g min. 20 fractions of 2 ml each were
collected from bottom to top. Two peroxisomal peaks were obtained with
densities of 1.24-1.23 g/cm (fractions 2 and 3
highly purified ``heavy'' peroxisomes) and
1.15-1.14 g/cm
(fractions 15 and 16
``light'' peroxisomes). The light peroxisomal fraction
contained mainly microsomes as well as small PO with high levels of
-oxidation activity. This fraction corresponds to the one recently
characterized by Schrader et al.(24) in human
hepatoblastoma (HepG2) cells, which was also obtained by density
gradient centrifugation using an exponential metrizamide gradient. -hydroxyacid oxidase(25) , palmitoyl-CoA
oxidase(26) , enoyl-CoA hydratase(27) ,
cyanide-insensitive palmitoyl-CoA -oxidation (28) phosphoglucomutase, phosphoglucoisomerase, and
glyceraldehyde-3-phosphate dehydrogenase(29) ,
esterase(30) , and cytochrome-c oxidase(31) .
LDH and acid phosphatase were assayed using commercially available
test-kits(53) . Protein was measured according to Lowry et
al.(32) with bovine serum albumin as standard. Data are
presented in histogram form(30) .Subfractionation of Isolated Peroxisomes
The
peroxisomal matrix proteins were extracted with a hypotonic TVBE buffer
(0.01% Triton X-100, 0.1% ethanol, 1 mM NaHCO, 1
mM EDTA, pH 7.6) followed by separation of the core and
membranes by centrifugation for 60 min at 100,000 g.
For the isolation of the peroxisomal integral membrane proteins, PO
were treated with an alkaline carbonate buffer, pH 11.5, according to
Fujiki et al.(33) . The cores were purified by
metrizamide density gradient centrifugation(34) .SDS-PAGE and Western Blotting
Polypeptides of
highly purified PO, PO subfractions, and cytosolic fractions were
separated by SDS-PAGE (10% resolving mini slabgels, 8 4.5
0.1 cm) and were either silver-stained or electrotransferred to
nitrocellulose membranes (22) . For immuncomplexing, the
monospecific polyclonal rabbit anti-rat-LDH-antibody was used. The
antibodies against rat urate oxidase and catalase were raised in
rabbits and characterized previously(34, 35) . The
antibody to the rat 22-kDa peroxisomal membrane protein (PMP 22) was
kindly provided by Professor Hashimoto (Shinshu University, Matsumoto,
Japan). The immune complexes were visualized by a modified protein
A-gold technique(35) .Isoelectric Focussing (IEF)
IEF polyacrylamide
gels with a linear gradient, pH 2-11, were run on a Multiphor
system according to the instructions of the manufacturer, Pharmacia
Biotech, Inc. For crude peroxisomal and cytosolic fractions, amounts of
protein corresponding to 5 milliunits of LDH activity were loaded per
lane. The isoforms of LDH in the subcellular fractions were visualized
by staining of the enzyme activity with the nitro blue tetrazolium
method(4) , as well as by immunoblotting with a modified
protein A-gold procedure(35) .Differential Salt Extraction of Isolated
Peroxisomes
Freshly isolated PO (0.668 mg/ml) were diluted
10-fold in 5 mM MOPS, pH 7.4, 1 mM EDTA, 0.05%
deoxycholate containing different concentrations of KCl (20, 100, and
500 mM). After incubation for 15 min on ice, the mixtures were
centrifuged for 60 min at 100,000 g, and pellets and
supernatants were assayed for protein, LDH, and catalase activities.Limited Proteolysis of Isolated
Peroxisomes
Freshly isolated PO were diluted (1:10) either in
isotonic homogenization buffer (5 mM MOPS, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1% ethanol) or hypotonic TVBE
buffer (see above) and subjected to repeated freeze/thaw cycles (4
times each). The appropriate preparations were treated with proteinase
K (1 mg/ml stock solution in 50 mM Tris buffer, pH 8; final
concentration, 0.33 mg/ml) for different time intervals up to 60 min at
room temperature. The reaction was stopped by the addition of
phenylmethylsulfonyl fluoride (0.2 mM final concentration). As
controls, freshly isolated unfrozen PO were diluted in the gradient
medium with appropriate banding density and treated in parallel. The
enzyme activities of catalase and LDH were determined, and the results
were presented as a percentage of the corresponding total activities. ; final concentration, 0.33 mg/ml) for different time
intervals up to 60 min and subjected to IEF, followed by activity
staining. For this purpose, (a) the undiluted unfrozen PO were
treated with Triton X-100 (0.2 and 1%) before protease digestion and (b) metrizamide was added to the cytosolic fraction in a
concentration corresponding to that in PO gradient fractions in order
to rule out any influence of the high metrizamide concentration in
peroxisomal fractions on proteolysis.Peroxisomal Palmitoyl-CoA-
LDH and cyanide-insensitive Oxidation and LDH
Activity-oxidation of
palmitoyl-CoA (28) were measured in freshly isolated hepatic PO
of control rats and animals treated for 7 and 14 days with 75 mg/kg
bezafibrate. In order to evaluate the impact of LDH activity on
peroxisomal -oxidation, the cyanide-insensitive palmitoyl-CoA
-oxidation was assayed in the presence of (a) different
concentrations of pyruvate (0.1-20 mM), (b)
increasing concentrations of the LDH-inhibitor oxamic acid
(0.05-10 mM) in the presence of 2 mM pyruvate,
and (c) -ketoacids other than pyruvate, glyoxylate, and
oxaloacetate (2 mM). Moreover, the activities of the
individual enzymes of the -oxidation system, i.e. palmitoyl-CoA oxidase (26) and enoyl-CoA
hydratase(27) , were determined in the presence of 0.1-20
mM pyruvate.Immunoelectron Microscopy
The livers of all of the
animals were fixed for 5 min by perfusion via the portal vein with a
fixative containing 0.25% glutaraldehyde, 2% sucrose, and 0.1 M PIPES buffer, pH 7.4. The tissue was processed for immunoelectron
microscopy as described previously(36) . Embedding of isolated
peroxisomal fractions and postembedding protein A-gold
immunocytochemistry was done according to Baumgart(37) .
Silver-intensification of the gold particles was accomplished in a
light-tight box using a slight modification of the method described by
Danscher et al.(38) .
Association of LDH with Highly Purified
Peroxisomes
After the differential centrifugation, the marker
enzymes of PO (particulate catalase and -hydroxyacid oxidase) were
associated mainly (>23%) with the L fraction (Fig. 1),
mitochondrial cytochrome-c oxidase was associated with the M
fraction (>80%), and microsomal esterase (>73%) was associated
with the P fraction (data not shown). The bulk of LDH activity was
recovered in the cytosolic fraction (S), with only about 1% of the
total activity being present in the crude peroxisomal fraction (Fig. 1). After density gradient centrifugation for the
isolation of highly purified PO, however, LDH consistently colocalized
with peroxisomal marker enzymes (Fig. 2). As further shown in Fig. 2, the main portion of particle-bound LDH activity was
associated with the highly purified heavy peroxisomal fractions 2 and
3, ( = 1.23-1.24 g/cm
) as well as with
the fractions 15 and 16 with lower densities ( =
1.13-1.14 g/cm
). The latter contained in addition to
microsomal proteins as measured by esterase activity (data not
presented), the ``light'' PO, as shown by peroxisomal
palmitoyl-CoA oxidase distribution. Interestingly, the PO in the heavy
and light fractions differed substantially in their enzyme composition,
with the light fractions containing significantly higher ratios of
palmitoyl-CoA oxidase/catalase (factor 5). These results are
consistent with the data reported previously by Schrader et
al. (24) from our laboratory and confirmed recently by
Wilcke et al.(39) . In contrast, other cytosolic
enzymes involved in glycolysis, phosphoglucomutase,
phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase
were not detectable in the highly purified peroxisomal fractions (Table 1). This would argue against a nonspecific uptake of
cytosolic LDH into peroxisomes. Subfractionation of purified
peroxisomal fractions followed by Western blotting using an anti-LDH
antibody revealed the association of LDH with the soluble peroxisomal
matrix proteins (Fig. 3), whereas the core and membrane
fractions were negative.
-HAOx), and LDH)
after subcellular fractionation by differential centrifugation
according to Völkl and Fahimi(18) . The
rates of recovery of marker enzymes were as follows: catalase: M, 20%;
L, 23%; P, 17%; S, 39%; glycolate oxidase (-HAOx): M,
15%; L, 23%; P, 17%; S, 43%; LDH: M, 9%; L, 0.5%; P, 17%; S, 73%.
Abbreviations used are as follows: M, heavy mitochondria; L, light mitochondria crude PO; P, microsomes; S, cytosol; U, total units of an enzyme found in a
single fraction;
U, total units found in all fractions,
p = total protein content of a single fraction,
p = total protein content of all
fractions.
-HAOX)) after further purification of the L-fraction by Metrizamide density gradient centrifugation. In
addition to the marker enzymes presented on the graph, the ones for
other cell organelles were enriched in the following fractions:
cytochrome-c oxidase (mitochondria), fractions 13 and 14;
esterase (microsomes), fractions 15 and 16; acid phosphatase
(lysosomes), fractions 17 and 18. U, total units of an enzyme
found in a single fraction; U, total units found in all
fractions;
V, total volume of a single fraction; V, total volume of all fractions; /////, heavy PO
(fractions 2 and 3) banding at = 1.23-1.24
g/cm
; , light PO (fractions 15 and 16) banding at
= 1.13-1.14
g/cm
.
-lactoglobulin.
Peroxisomal and Cytosolic LDHs Are Closely Related
Proteins
Since the total activities of LDH in cytosolic and
peroxisomal fractions differed markedly, aliquots of each fraction
exhibiting equal LDH-activities were used for Western blot and IEF
analysis. As shown in Fig. 4A, both polypeptides have
the same molecular weight (M
35,000).
Additionally, IEF revealed that both LDH-A- and
LDH-AB-isoforms are present in PO (Fig. 4B). Interestingly, the proportion of
LDH-AB is higher in PO than in the cytosol.
and LDH-AB
isoforms in both subcellular compartments. Protein amounts
corresponding to 5 milliunits of LDH activity were loaded per lane:
cytosol (C), 1.5 µg; crude peroxisomes (P), 38
µg. Molecular mass standards in kDa are as follows: 45,
ovalbumin; 24, trypsinogen; 18.5,
-lactoglobulin.
LDH Is a Bona Fide Peroxisomal Protein
To assess
the extent of peripheral association of cytosolic LDH to the surface of
PO, freshly isolated peroxisomal fractions were subjected to salt
extraction with increasing KCl concentrations (Fig. 5) and to
limited proteolysis ( Fig. 6and 7). As shown in Fig. 5,
the extraction patterns of LDH and catalase were comparable, confirming
the intraperoxisomal localization of LDH. The release of about 40% of
peroxisomal catalase in the absence of KCl is due to the dilution of
the peroxisomal fraction in the hypotonic buffer and subsequent
centrifugation at 100,000 g in this experiment. In Fig. 6, the rates of degradation of LDH and catalase in
differently pretreated PO are compared. Whereas in repeatedly frozen
and thawed peroxisomes (diluted in an isotonic homogenization buffer)
the degradation rates of both enzymes were comparable, in intact PO,
major differences were observed. The initial increase of catalase
activity after 15 min of proteolysis under the latter conditions is
most probably due to a better accessibility of the enzyme to its
substrate H
O. Whereas LDH activity was reduced
to 60% after 60 min of digestion, the catalase activity was only
moderately affected. This suggests that the effect on LDH could be due
in part to the removal of LDH associated with the cytosolic surface of
peroxisomes. After complete lysis of PO by freeze-thawing in hypotonic
buffer, the activity of both enzymes was completely abolished after 30
min of protease digestion.
g, and pellets and
supernatants were assayed for protein, LDH, and catalase activities.
The results are expressed as the release in percent of total activity
or protein.
], 
, intact PO diluted in
gradient medium for the assay; ,
, frozen/thawed
(4) PO in isotonic homogenization buffer; ,
,
frozen/thawed (4) PO in hypotonic TVBE
buffer.
Immunoelectron Microscopic Localization of LDH in
Peroxisomes: Evidence for a Heterogeneous Distribution in the
Peroxisomal Population
Postembedding protein A-gold
immunocytochemistry of liver sections revealed a predominantly
cytosolic localization of LDH. In addition, a heterogeneous labeling of
PO is observed, with most of the PO exhibiting only a few gold
particles in their matrix. A selected area of a liver cell is shown in Fig. 8a, where the PO are intensively labeled with gold
particles representing LDH antigen. The membranes and cores are not
stained in these organelles, whereas the peroxisomal matrix is strongly
labeled. Other cell organelles such as mitochondria, lysosomes, and ER
are negative (Fig. 8b). As controls for the specificity of
the immunocytochemical detection of LDH, liver sections were incubated
in parallel with an anti-catalase antibody, which revealed exclusive
peroxisomal labeling (Fig. 8c) or with an appropriate
LDH-preimmune serum, which showed no labeling (Fig. 8d).
Peroxisomal LDH Is Increased by Bezafibrate
Treatment
In Table 2the activities of palmitoyl-CoA
oxidase and LDH in total homogenates and highly purified PO obtained
from livers of rats treated for 7 and 14 days with bezafibrate are
compared with those of control animals. Whereas the palmitoyl-CoA
oxidase activity in total homogenates was increased up to 9-fold by the
treatment, that of LDH was only slightly elevated. In highly purified
peroxisomal fractions on the other hand, the elevation of LDH activity
was much more pronounced than in homogenates, reflecting a selective
induction of the peroxisomal LDH. Similarly, in Western blots the
amount of LDH protein was significantly increased in peroxisomal
fractions of bezafibrate-treated animals (Fig. 10).
-lactoglobulin.
NADH Provided by Peroxisomal
The cyanide-insensitive
-Oxidation Is
Reoxidized by LDH in Peroxisomes-oxidation system (5 mM palmitoyl-CoA, 100 mM KCN, 37 °C) of highly purified PO of bezafibrate-treated
animals (75 mg/kg/day) produced 179.7 nmol of NADH/min/mg of protein.
Under similar assay conditions (2 mM pyruvate, 100 mM KCN, 37 °C), the peroxisomal LDH oxidized 930 nmol of
NADH/min/mg of protein, suggesting that it is capable to reoxidize
completely the NADH produced by the peroxisomal -oxdation system. -oxidation is
summarized. Table 3shows that the degree of reoxidation of NADH
was clearly dependent on the concentration of pyruvate added to the
mixture used for assaying the -oxidation activity. Reoxidation of
NADH is maximal at 2 mM pyruvate, a concentration that results
in optimal LDH rates and is diminished at higher pyruvate
concentrations known to inhibit LDH activity. Thus, it seems that NADH
produced in PO is reoxidized indeed by intraperoxisomal LDH. This
notion is further supported by the data presented in Table 4. A
dose-dependent inhibition of NADH-reoxidation was noted with increasing
concentrations of oxamic acid, an inhibitor of LDH. In Table 5,
the production rates of NADH by the peroxisomal -oxidation system
in the presence of pyruvate and other -ketoacids are compared.
Even though glyoxylate can be converted by LDH to oxalate, 2 mM glyoxylate in the -oxidation assay mixture exerted no effects
on NADH production rates. Only at higher concentrations (5
mM), 36.4% of the NADH produced was reoxidized. These data are
consistent with 470 times higher K
values of
peroxisomal LDH for glyoxylate compared with pyruvate (K-glyoxylate: 5.83 10 mol/liter; K
-pyruvate: 1.24
10 mol/liter). On the other hand, oxaloacetate
proved to be almost as effective as pyruvate, suggesting the presence
of an additional dehydrogenase (possibly malate dehydrogenase) in
peroxisomes.
-oxidation
system present in this cell organelle.LDH Is Associated with Different Cell Organelles
An
association of LDH with different cell organelles such as the nucleus,
mitochondria or microsomes was proposed already in the sixties by
Agostini et al.(40) . Since then the debate on the
subcellular distribution of LDH has continued mainly because the
LDH-isoforms found in the different cell compartments were similar and
the percentage of total activity associated with the different
organelles was very low (e.g. 1% for nuclear and
1-1.6% for mitochondrial
LDH)(6, 9, 10) . In the meantime, only the
nuclear localization of LDH has been confirmed by immunoelectron
microscopy(41) . In addition, this nuclear enzyme was shown to
be posttranslationally modified by the phosphorylation of the tyrosine
residue 238 and to behave like a single-stranded DNA-binding protein (6) .
) described by McGroarty et al.(10) was identical to that of the cytosolic fraction and
their kinetic properties were similar, it has since been generally
concluded that the LDH in peroxisomal fractions is a cytosolic
contaminant(11) . = 1.23-1.24
g/cm
) at the bottom of the gradient, whereas the second
peak was associated with the microsomal fractions (fractions 15 and 16;
= 1.13-1.14 g/cm
) at the top of the
gradient immediately below the soluble components containing the so
called 178 light peroxisomes 178 (Fig. 2). Recently, Schrader et al.(24) have demonstrated, that this second peak
with low density contains a large number of small PO that exhibit a
relatively high ratio of -oxidation enzymes to catalase. Moreover,
Wilcke et al.(39) demonstrated by postembedding
immunocytochemistry of the low density fractions obtained from
di(ethylhexyl)phthalate-treated animals, that indeed the small
peroxisomal vesicles present in these fractions contain significant
amounts of -oxidation enzymes.LDH Is a Bona Fide Peroxisomal Matrix Enzyme and Is
Distributed Heterogeneously in the Peroxisomal Population
Since
it was reported that all LDH-isoforms present in different cell
organelles in hepatocytes resembled the cytosolic ones(40) , we
decided to use the isolated cytosolic LDH for generation of a rabbit
anti-rat liver-LDH antibody. After confirmation of the monospecificity
of our antibody for LDH, it was used for the immunocytochemical
detection of the LDH protein in rat liver sections. The results clearly
indicate the presence of LDH in the peroxisomal matrix, in addition to
the staining of the cytoplasm and the nucleus, whereas the remainder of
the cell organelles such as mitochondria, lysosomes, or ER appeared
negative (see Fig. 9, a and b). In addition,
the morphological data provide strong evidence for a heterogeneous
distribution of the LDH in PO. These results are in full agreement with
those obtained after subfractionation of PO, where both LDH activity
and LDH protein detected by Western blotting were found in the matrix
fraction of PO. Although the isoenzyme composition of the cytosolic and
peroxisomal LDHs are very similar, as shown by IEF and gel
electrophoresis, highly purified PO contained relatively more
LDH-AB than the cytosolic fraction (Fig. 4B). This isoenzyme pattern was also confirmed by
blotting of the IEF-gels and immunodecoration of the blots for LDH
(data not shown). The LDH-AB band in PO became even more
prominent after mild ``proteinase K-stripping'' of intact PO (Fig. 7A, compare P
with
P
-P).
At Least 80% of the LDH Activity in Isolated Peroxisomal
Fractions Is Truly Intraperoxisomal
As shown by selective salt
extraction, limited proteolysis of intact and partially extracted PO
and separately by immunoelectron microscopy, approximately 10-20%
of the peroxisomal LDH is bound to the cytosolic surface of the
peroxisomal membrane. Even though only 60% of the LDH activity is
retained after 60 min of protease treatment of freshly isolated intact
PO (Fig. 6), a higher intraperoxisomal LDH-percentage can be
assumed, since after this time period, the catalase activity was
reduced by about 25% also. Additional support for the presence of more
than 80% of peroxisomal LDH activity being in the matrix is provided by
the kinetics of proteolytic degradation of LDH as shown by IEF (Fig. 7A). Whereas the cytosolic LDH was completely
degraded after 15 min of protease treatment, that of intact PO
persisted more than 30 min and indeed made up to 80% of the total
activity. Most convincingly, quantitative analysis of immunoelectron
microscopic preparations of isolated peroxisomal fractions showed that
about 20% of the gold particles are attached to either side of the
peroxisomal membrane with 80% being localized in the matrix (Fig. 9c).Peroxisomal LDH Is Coupled to the
Whereas the presence of
several NAD-Oxidation System
and Reoxidation of NADH in Peroxisomes-linked dehydrogenases, such as
-glycerol phosphate dehydrogenase(13) , alcohol
dehydrogenase(14) , and different 3-hydroxyacyl-CoA
dehydrogenases (12) has been well established in mammalian PO,
no NADH-reoxidizing system has been described in this cell organelle.
Since the peroxisomal membrane seems to be permeable in vitro for small solutes and coenzymes(15) , the reoxidation of
NADH in the cytosol after passage across the peroxisomal membrane has
been considered and discussed(11) . In view of the clear
compartmentation and strict regulation of cytoslic and mitochondrial
NAD/NADH-pools by the malate-aspartate,
-glycerolphosphate, and malate-pyruvate shuttle
systems(42) , it seemed very unlikely to us that a
membrane-bounded organelle such as the PO should be permeable under in vivo conditions to these cofactors. In plants a
malate-oxaloacetate-aspartate shuttle between glyoxysomes,
chloroplasts, and mitochondria during the photosynthetic respiration
has been envisaged with glyoxysomal malate dehydrogenase being the
enzyme reoxidizing the reduced NADH in this cell organelle (43, 44) . Finally, at the time of preparation of this
manuscript van Roermund et al. (16) reported that PO
in S. cerevisiae are not permeable to
NAD/NADH in vivo and that a malate
dehydrogenase-linked shuttle system is present in them. Support for the
coupling of LDH to the
-oxidation system and the reoxidation of
NADH in mammalian PO is provided by the following lines of evidence: (a) peroxisomal fractions of animals treated with bezafibrate
exhibited higher levels of peroxisomal LDH activity (Table 2) and
contained higher amounts of LDH protein (Fig. 10), (b)
in highly purified peroxisomal fractions from obese mice containing
enhanced levels of -oxidation enzymes also elevated LDH-levels
were reported(45) , and (c) pyruvate stimulated the
-oxidation of palmitoyl-CoA and erucoyl-CoA in peroxisomal
fractions, whereas the addition of exogenous LDH did not lead to
further stimulation(17) .-oxidation of palmitoyl-CoA was
demonstrated (Table 3-V). Thus, NADH-production rates
measured in the -oxidation assay were inversely proportional to
the LDH activity in PO (Table 3), with NADH reoxidation rates
being maximal at maximal LDH activity (2 mM pyruvate).
Inhibition of peroxisomal LDH by high pyruvate levels (up to 20
mM) or by the addition of oxamic acid restored NADH production
rates (Table 4). A direct inhibition of palmitoyl-CoA oxidase or
3-enoyl-CoA hydratase by pyruvate leading to changes in NADH production
could be excluded in our study by measuring the enzymes separately in
the presence of increasing amounts of pyruvate (data not shown). Thus,
the data demonstrate that peroxisomal LDH is capable of reoxidizing
NADH generated in the PO and suggest that LDH may play a role in
regulating the peroxisomal NAD/NADH ratio and in
maintaining the flux of fatty acids through the peroxisomal
-oxidation system./NADH levels, one has
to assume that the peroxisomal membrane in vivo displays a
restricted permeability to these cofactors and that LDH constitutes a
component of a shuttle system transferring reducing equivalents across
the peroxisomal membrane. Fig. 11depicts such a putative
shuttle mechanism. Lactate generated inside the PO by the action of
peroxisomal LDH crosses the peroxisomal membrane and is reoxidized in
the cytosol to pyruvate, which reenters the PO, thus resulting in the
transfer of the reducing equivalents from the PO to the cytosol.
-oxidation
spiral; 1, activated long chain fatty acid; 2,
3-hydroxyacyl-CoA; 3, 3-ketoacyl-CoA; 3-OH-DH,
3-hydroxyacyl-CoA dehydrogenase; pLDH, peroxisomal LDH; cLDH, cytoplasmic LDH.
and AB oligomers
into peroxisomes.
)
We thank A. Achten, I. Frommer, G.
Krämer, H. Mohr, and K. Rummer for skillful
technical assistance and secretarial help. We also thank Professor G.
Mannaerts, University of Leuven, Belgium, for critical review of the
manuscript and helpful comments and Professor T. Hashimoto for
providing the antibody to PMP 22.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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