ARTICLE |
Correspondence to: Sadaki Yokota, Biology Laboratory, Yamanashi Medical University, Tamaho-cho, Yamanashi, 409-3898, Japan. E-mail: syokota@res.yamanashi-med.ac.jp
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Summary |
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We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.
(J Histochem Cytochem 51:215226, 2003)
Key Words: NADP-dependent isocitrate, dehydrogenase, mitochondria, nuclei, immunoelectron microscopy
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Introduction |
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IN EUKARYOTES, three different isocitrate dehydrogenases (ICDs) catalyze the decarboxylation of isocitrate into 2-oxoglutarate. NAD-dependent ICD (EC 1.1.1.41) is present in mitochondria, while NADP-dependent ICDs (EC.1.1.1.42) are found in mitochondria (ICD1) and in the cytosol or peroxisomes (ICD2). These enzymes catalyze the following reversible reaction:
Isocitrate + NADP + 2H+ 2-oxoglutarate + NADPH + H+ + CO2
NAD-dependent isocitrate dehydrogenase catalyzes a key step in the tricarboxylic acid cycle, whereas the physiological roles of ICD1 and ICD2 are not clearly understood. Both enzymes have been purified from various sources and their cDNA sequences determined (
The amino acid sequence of ICD1 has been determined in several animals, plants, and yeasts (
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Materials and Methods |
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Animals
Male Wistar rats weighing 180200 g and Japanese White rabbits weighing 34 kg were fed on standard diets for each animal and water ad libitum until use. The animal experiments were performed in accordance with the Guidance for Animal Experiments, Yamanashi Medical University.
Antibodies
Anti-ICD1 peptide antibody was prepared as follows. A peptide consisting of 14 amino acids (DTIKSNLDRALGKQ) at the C-terminus of mouse ICD1 was synthesized and cysteine was added at the N-terminus. One milligram of synthetic peptide was conjugated to 1.6 mg of keyhole limpet hemocyanin (Sigma; St Louis, MO) with MBS. Two milliliters of conjugate containing 300 µg of peptide was emulsified with the same volume of complete Freund adjuvant and the emulsion was injected into the back of two Japanese White rabbits. Every 2 weeks each rabbit was immunized with 150 µg of peptide and after 8 weeks blood was collected from an ear vein. Reactivity was tested by dot-blotting using peptide-conjugated BSA. Rabbit and guinea pig anti-rat liver catalase antibodies were prepared as described previously (
Reactivity Test of Anti-ICD1 Peptide Antibody
ICD Activity in Heart Extract After Incubation with Anti-ICD1 Peptide Antibody-absorbing PVDF Membrane.
Specific antibody to ICD1 peptide was isolated as follows. PVDF membrane was incubated in a solution of ICD1 peptide-conjugated BSA (1 mg peptide/ml). Having absorbed 0.672 mg of peptide, it was then incubated with anti-ICD1 anti-sera for 2 hr at room temperature (RT). Specific antibody binding to peptide-BSA was eluted with 0.1 M glycine-HCl buffer (pH 2.3) containing 0.5 M NaCl at 0C and the eluate was quickly neutralized with 1 M Tris. The absorption and elution were repeated and the eluates were pooled. Specific antibody was absorbed by a new PVDF membrane (4 cm2). The membrane that had absorbed specific ICD1 peptide antibody was soaked in 0.5% gelatinPBS and cut into pieces of 5 mm x 5 mm. Ten percent rat heart homogenate was extracted with the same volume of 2% Triton X-100 in 50 mM potassium phosphate buffer (pH 7.4) and the extract was diluted fivefold. Each 100 µl of extract was incubated with 0, 1, 2, 4, and 6 pieces of antibody-PVDF membrane, respectively, for 3 hr at 4C. For the control experiment, a number of PVDF membrane pieces absorbing IgG from pre-immune serum were incubated with the heart extract. Then the ICD1 activity of the extracts was assayed by the method of
Western Blotting Analysis of Anti-ICD1 Antibody.
Heart and kidney of rat and mouse were homogenized in a solution consisting of 0.1 µM potassium phosphate buffer (pH 7.4), 0.1% Triton X-100, 10 µM PMSF, 4 µM leupeptin, 4 µM chymostatin, 4 µM antipain, and 4 µM pepstatin using a HG30 homogenizer (Hitachi; Tokyo, Japan). The homogenates were centrifuged at 100,000 x g for 1 h using a 60Ti rotor (Beckman Japan; Tokyo, Japan). The resulting supernatants were used for SDS electrophoresis (
Immunoblotting Analysis of Tissue Homogenates
Anesthetized rats were decapitated and the submandibular gland, heart, lung, liver, small intestine, colon, diaphragm, spleen, adrenal gland, kidney, testis, epididymis, cerebrum, cerebellum, and brown fat were dissected. Each organ was homogenized in a homogenizing medium containing 50 mM potassium phosphate buffer (pH 7.4), 1% Triton X-100, 10 µM PMSF, 4 µM leupeptin, 4 µM chymostatin, 4 µM antipain, and 4 µM pepstatin. The concentration of protein was adjusted to 1 mg/ml. Homogenates were mixed with the same volume of sample buffer for SDS-PAGE and heated in boiling water for 2 min. Five micrograms of sample were loaded on the gels and electrophoresis was carried out. After electrophoresis, proteins were transferred onto PVDF membranes (Millipore; Bedford, MA) and stained with a combination of HRP-labeled goat anti-rabbit IgG and DAB.
Nycodenz Gradient Centrifugation
A light mitochondrial fraction was prepared from rat liver homogenate as described above. According to the method described by
Isolation of Cell Nuclei
The method described by
Other Analytical Procedures
SDS-PAGE was carried out according to
Immunocytochemical Procedures
Immunofluorescence Microscopy.
Rats were anesthetized with ether. The heart, liver, and kidneys were fixed by perfusion through the left ventricle, portal vein, and abdominal aorta, respectively, for 10 min at RT. The fixative consisted of 4% paraformaldehyde, 0.2% glutaraldehyde, and 0.2 M HEPES-KOH buffer (pH 7.4). Small tissue blocks were further fixed in 1% glutaraldehyde buffered with 0.1 M HEPES-KOH (pH 7.4) for 1 hr at 4C. After being washed in PBS, tissue blocks were dehydrated in graded ethanol and embedded in Epon. Polymerization of Epon was performed overnight at 60C. The staining method used has been described previously (
Postembedding Immunoelectron Microscopy
Preparation of Protein AGold and IgGGold Probes
Two kinds of colloidal gold (15 nm and 8.5 nm in diameter) were prepared by the method of
Tissue Preparation and Immunostaining Small tissue slices of rat heart, liver, and kidney fixed by perfusion with 4% paraformaldehyde + 0.2% glutaraldehyde were dehydrated with graded ethanol at -20C and embedded in Lowicryl K4M or LR White. Polymerization of the resins was performed under UV light overnight at 20C. Ultrathin sections were cut with a diamond knife using a Reichert Ultracut R and mounted on nickel grids. The sections were incubated with 500-fold diluted anti-ICD1 antibody overnight at 4C, followed by a 30-min incubation with protein Agold probe (15 nm). Some sections were stained doubly by a combination of anti-ICD1 and anti-rat liver catalase with 8-nm or 15-nm protein Agold probes. The sections were electron stained with uranyl acetate for 10 min and with lead citrate for 30 sec. All thin sections were examined with a Hitachi H7500 electron microscope at an accelerating voltage of 80 kV.
Quantitative Analysis of Gold Labeling Ten digital micrographs were taken from postembedding immunoelectron microscopic sections at a magnification of x15,000 and printed out by a laser printer. Areas of mitochondria, peroxisomes, nuclei, and cytoplasm were determined by a digitizer equipped with a computer and the gold particles present in each area were counted. Labeling density was expressed as gold particles/µm2.
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Results |
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Reactivity of Anti-ICD1 Peptide Antibody with Peptide
ICD Activity in Heart Extract After Incubation with PVDF Membrane Absorbing Anti-ICD1 Peptide Antibody.
We tested whether the antibody can remove ICD activity from tissue extract using small pieces of antibody-absorbing PVDF membrane. When a fixed amount of rat heart extract was incubated with a number of membrane pieces, the ICD activity in the extract was found to decrease as the number of membrane pieces increased (Fig 1, open circles). However, PVDF membrane absorbing IgG from pre-immune serum did not change the ICD activity in the extract (Fig 1, closed circles). The results clearly showed that our antibody to the 14-amino-acid peptide of the mouse ICD1 C-terminus could bind to rat heart ICD1.
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Immunoblotting Analysis of Anti-ICD1 Antibody. The present antibodies were raised in two rabbits immunized with 14 amino acids at the C-terminus of mouse ICD1. The amino acid sequence at the C-terminus of rat ICD1 has not yet been determined. Then we tested whether the anti-ICD1 peptide antibody could react with heart and kidney extracts of rat by Western blotting. Both antibodies recognized a single apparently identical band in the rat heart and kidney extracts (Fig 2). The reactivity of both antibodies was very similar, and we used them in subsequent experiments. The molecular mass of the band was calculated to be approximately 45 kD.
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Immunoblot Analysis of Expression of ICD1 in Various Tissues
We analyzed 15 different tissues, i.e., salivary gland, liver, small intestine, colon, spleen, heart, diaphragm, adrenal gland, kidney, testis, epididymis, lung, cerebrum, cerebellum, and brown fat, from male Wistar rats. Among these samples, the highest level of ICD1 was expressed in heart muscle, followed by kidney and brown fat (Fig 3). In other tissues, a low level of ICD1 was expressed. No signals were detected in testis at the protein concentration (1 mg/ml) used. In most tissues examined, a single band was observed and the molecular mass was calculated to be approximately 45 kD. In cerebrum, a minor band of 45 kD and major band of 90 kD, which seemed to be a dimer of the 45-kD subunit, were detected (not shown).
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Localization of ICD1 in Rat Liver After Nycodenz Gradient Centrifugation
We subjected the light mitochondrial fractions of rat liver to Nycodenz gradient centrifugation. The results are shown in Fig 4. The ICD1 antigen, which had the same molecular weight as that shown in Fig 2, was detected in fraction numbers 2024, in which a mitochondrial marker antigen, acy-CoA dehydrogenase, was also detected. In peroxisomal fractions (fraction numbers 35), catalase antigen was detected, whereas ICD1 antigen was not. The results clearly showed that, in rat liver, ICD1 was exclusively distributed in the mitochondria. We have tried to isolate peroxisomes from the light mitochondrial fraction of rat kidney and heart by the same technique, but we could not recover intact peroxisomes from the fractions.
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Immunolocalization of ICD1 in Rat Heart and Kidney
Immunofluorescence Staining of ICD1
Heart.
We used deplasticized semithin sections of Epon-embedded tissues for immunofluorescence staining (
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Kidney Next we stained Epon sections of rat kidney after removal of epoxy resin. Cytoplasmic granules were stained with variable intensity and nuclei of tubule epithelial cells and connective tissue were also weakly stained (Fig 6a). At higher magnification, the stained granules exhibited a typical mitochondrial profile (Fig 6b), which was proved by staining of a mitochondrial marker enzyme, 3-keto-acyl-CoA thiolase (Fig 6c). In the segments of renal tubules, there were two types. One essentially contained a few mitochondria and the other involved many mitochondria that were weakly stained or almost negative. Next, we stained serial sections for ICD1 and catalase to clarify whether ICD1 was localized in peroxisomes. The staining pattern for ICD1 was quite different from that for catalase (Fig 6d and Fig 6e). It appear that the same granules were not positive for both antigens. In some segments, mitochondria were heavily stained for ICD1 but did not appear to be stained for catalase (Fig 6d and Fig 6e). Cell nuclei in renal tubules as well as connective tissue were stained (Fig 6a). The staining intensity of nuclei was essentially lower than that of mitochondria. Therefore, when sections became thinner than 100 nm, the nuclear staining was indistinguishable (Fig 6d and Fig 6e).
Immunoelectron Microscopy. Gold labeling for ICD1 was stronger in Lowicryl K4M-embedded than in LR White-embedded materials. The immunoelectron microscopic data shown here were all obtained from the materials embedded in Lowicryl K4M.
Heart Gold particles showing subcellular sites of ICD1 were almost exclusively confined to mitochondria and cell nuclei, being absent from sarcoplasmic reticulum and myofibrils (Fig 7a and Fig 7b). In the nuclei, gold particles were closely associated with heterochromatin and nucleolus (Fig 7a and inset). When sections were stained doubly for ICD1 and catalase using different-sized gold probes, ICD1 antigenic sites visualized with a small gold probe (8.5 nm in diameter) were found in the mitochondria, whereas catalase shown by a large gold probe (15 nm in diameter) was found exclusively in peroxisomes (Fig 7c and Fig 7d). In control sections incubated with pre-immune serum instead of anti-ICD1 peptide antibody, followed by protein Agold probe, no specific labeling in mitochondria was noted (data not shown).
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Kidney Signals of ICD1 were observed in mitochondria and cell nuclei but not in peroxisomes and other organelles (Fig 8a and Fig 8b). The staining was much weaker in the nucleus than in mitochondria. In the nucleus, most gold particles were associated with heterochromatin and nucleolus (Fig 8a and inset). As shown by immunofluorescence staining, the staining intensity varied among the segments of renal tubules. The highest staining intensity was noted in particular segments of distal tubules, in which only a few peroxisomes were present (Fig 8b). Except for the S3 segment, mitochondria of proximal tubules and collecting tubules generally exhibited very low staining intensity. The labeling intensity was essentially lower than that in heart muscle mitochondria. After the double labeling of ICD1 and catalase with protein Agold probes of different sizes, no ICD1 was detected in peroxisomes (Fig 8c and Fig 8d). In immunocytochemical control sections, no specific labeling was noted in mitochondria and nuclei (data not shown).
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Quantitative Analysis of the Gold Labeling Next, we analyzed the labeling density in heart muscle and kidney. The results are shown in Table 1. In both tissues, the labeling density of mitochondria and nuclei was significantly higher than that of cytoplasm. The labeling density of nuclei was approximately half the mitochondrial labeling density in the heart and kidney. The cytoplasmic labeling was similar to that obtained in immunocytochemical control experiments. The labeling density of heart muscle mitochondria and nuclei was approximately double that of kidney mitochondria and nuclei, respectively.
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Nuclear Localization of ICD1
Immunocytochemical staining showed that ICD1 is localized in cell nuclei. However, the nuclear fractions isolated from heart and kidney by differential centrifugation contained very low levels of ICD activity (data not shown). Then we isolated highly pure nuclei from rat kidney and liver by equilibrium sedimentation (
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Discussion |
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Specificity of Antibody
Rabbit antibody to a peptide of 14 amino acids at the C-terminus of mouse ICD1 was demonstrated to react with the peptide conjugated with BSA by dot-blotting analysis. Experiments using antibody-absorbing PVDF membranes showed that the anti-ICD1 peptide antibody could react with ICD1 to reduce the enzymatic activity in heart extract. Furthermore, Western blotting analysis showed that the anti-peptide antibody reacted with a single protein band with a molecular mass of 45 kD, which was considerably smaller than the unprocessed precursor form that had been predicted from DNA sequences so far: bovine, 50,824 (
Localization of ICD1 in Rat Liver After Nycodenz Gradient Centrifugation
We have tried to purify peroxisomes from the light mitochondrial fraction of rat kidney and heart by Nycodenz gradient centrifugation but we could not recover intact peroxisomes. Therefore, in the present study we subjected the light mitochondrial fractions of rat liver to Nycodenz gradient centrifugation. ICD1 was distributed in mitochondrial fractions where acyl-CoA dehydrogenase existed but not in peroxisomal fractions where catalase was detected. The results clearly show that ICD1 is a mitochondrial enzyme in the liver. The mitochondrial localization of ICD1 in heart and kidney was confirmed by immunocytochemical staining.
Localization of ICD1 in Heart and Kidney of Rat
Immunofluorescence microscopy of ICD1 showed that staining patterns were consistent with typical mitochondrial patterns seen in heart muscle and renal tubules. In heart muscles, staining of serial sections for ICD1 and catalase indicated that both enzymes were present in different cytoplasmic granules. In kidney, immunofluorescence for ICD1 varied in intensity among renal tubule segments. Strong staining was noted in mitochondria of the distal convoluted tubules, followed by the proximal tubules. Collecting tubules and the thin limb of the loop of Henle were stained only very weakly or not at all. We stained serial sections 100 nm thick for ICD1 and catalase. The results clearly showed that renal tubule segments heavily stained for ICD1 were hardly stained at all for catalase, whereas the segments moderately stained for ICD1 were strongly stained for catalase. It has been shown that in the kidneys most peroxisomes are located in the proximal but not the distal tubules (
Nuclear Localization of ICD1
In the present study, cell nuclei of heart and kidney were stained by both immunofluorescence and immunogold techniques. However, staining intensity of the nuclei was significantly weaker than that of mitochondria. Furthermore, the staining was not observed in the immunocytochemical control sections treated with pre-immune serum (data not shown). Immunogold staining showed similar results. The labeling density of mitochondria was approximately 10-fold that of nuclei in heart and about five-fold that in kidney. Biochemical assays of ICD activity in isolated nuclei also showed the presence of ICD1 in the nuclei, although the activity per mg of protein was essentially lower than in mitochondria. All together, the present results strongly suggest that ICD1 is present in cell nuclei of heart and kidney, including those of connective tissue and capillaries. It has been reported that no ICD activity was detected in nuclei isolated from rat liver and Zaidel's hepatoma by an enzyme cytochemical method (
In the present study, no apparent difference in molecular size was seen between mitochondrial and nuclear ICD1. This could be explained as follows. Newly synthesized ICD1 is imported to mitochondria, where its signal sequence is cut off, and then the mature ICD1 is exported to the cytoplasm. Finally, it is transported into the nucleus through an unknown mechanism. Recently, it has been reported that mitochondrion-resident proteins display function different from their original activity in the outside of mitochondria (
Physiological Function of ICD1 in Mitochondria and Nuclei
In the present study we have shown that the mitochondrial isoenzyme of ICD1 is localized in mitochondria as well as nuclei. In mitochondria there are two types of ICDs, NAD-specific and NADP-specific. Both enzymes catalyze oxidative decarboxylation of isocitrate to 2-oxoglutarate and require either NAD or NADP, producing NADH or NADPH, respectively. It has been shown that, in the yeast Saccharomyces cerevisiae, NAD-specific ICD significantly contributes to the tricarboxylic acid (TCA) cycle (
The present study has shown that ICD1 is present in the nuclei of various cell types, including epithelial cells of renal tubules, fibroblasts of connective tissues, endothelial cells of capillaries, and myocardial cells. The nuclei of some cells contained little or no stain. In the nuclei, immunofluorescence staining for ICD1 was dotted and appeared to be associated with chromatin. By IEM, gold particles were found predominantly attached to heterochromatin. These results suggest that nuclear ICD1 associates with heterochromatin but is not homogeneously scattered in the nucleoplasm. The biochemical role of nuclear ICD1 is not understood any better than that of mitochondrial ICD1. It is likely that nuclear ICD1 provides NADPH by its enzymatic oxidation of isocitrate or NADP by reduction of 2-oxoglutarate. NADP and NADPH are used as co-factors in various biochemical reactions. The nuclear metabolism of tirapazamine, a bioreductive drug now in clinical trials, is carried out by unknown reductases and is supported by NADPH or NADH (
In conclusion, we have demonstrated immunocytochemically that the major subcellular site for mitochondrial NADP-specific isocitrate dehydrogenase (ICD1) is in fact the mitochondria, and a minor site is nuclei in rat kidney and heart. In kidney, the mitochondrial content of ICD1 differs among the tubule segments, whereas in heart it is homogeneous. The nuclei of several cells and tissues contain ICD1, which is associated with heterochromatin.
Received for publication April 19, 2002; accepted September 6, 2002.
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