(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 > LDH-A
B)
with the peroxisomes comprising relatively more LDH-A
B 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.
Lactate dehydrogenase (L-lactate:NAD 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-A
B, 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) .
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 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 NAD
in
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) .
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 -oxidation system of this organelle is
demonstrated.
Enzyme activities were determined according to standard procedures:
catalase and -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) .
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; 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.
Figure 1:
Distribution of marker enzyme
activities (catalase, glycolate oxidase (-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.
Figure 2:
Distribution of marker enzyme activities
(catalase, palmitoyl-CoA oxidase (AOx), LDH, and glycolate
oxidase (-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
.
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,
-lactoglobulin.
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 and LDH-A
B
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.
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 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.
Figure 6:
Influence of different physical
pretreatment conditions on the accessibility of LDH and catalase in PO
as revealed by proteolysis. ],
, 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.
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,
-lactoglobulin.
In Table 3-V the influence of PO-associated LDH on the
reoxidation of NADH produced by peroxisomal -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.
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 -oxidation
system present in this cell organelle.
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) 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) .
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; = 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.
In a series of separate
experiments, a direct involvement of peroxisomal LDH in the reoxidation
of NADH produced by the -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.
If indeed the peroxisomal LDH plays a role
in regulating the peroxisomal NAD/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.
Figure 11:
Proposed shuttle mechanism for the
transfer of reducing equivalents between PO and cytoplasm. 1-3, intermediates of the -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.
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 and A
B oligomers
into peroxisomes.
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.