ARTICLE |
Correspondence to: Sadaki Yokota, Biological Laboratory, Yamanashi Medical University, Tamaho-cho, Yamanashi, 409-3898, Japan.
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Summary |
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Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:11231131, 2001)
Key Words: cytosolic NADP-dependent, isocitrate dehydrogenase, peroxisomes, Nycodenz gradient, centrifugation, immunoelectron microscopy
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Introduction |
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In mammals and yeast, three different isocitrate dehydrogenases (ICDs) that catalyze decarboxylation of isocitrate into -ketoglutarate are known. NAD-dependent ICD (EC 1.1.1.41) is localized in mitochondria. NADP-dependent ICD (ICD; EC.1.1.1.42) is found in mitochondria (ICD1) and in the cytosol (ICD2), and catalyzes the following reversible reaction:
Both isozymes have been purified from several mammals and their cDNA sequences were determined (
The peroxisomal membrane is very fragile, and therefore catalase, as well as other soluble enzymes, can leak out of peroxisomes into the cytosol during homogenization (
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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.
Purification of ICD2 from Rat Liver
ICD2 was purified from rat liver homogenate according to a slight modification of the method of
Antibodies
Rabbit antibody to ICD2 was prepared as follows. Two ml of purified ICD2 solution containing 4 mg was emulsified with the same volume of Freund's complete adjuvant. The emulsion containing 400 µg ICD2 was injected four times at intervals of 2 weeks into the back of Japanese White rabbits. Two weeks after the last injection, blood was collected and tested for immunoreactivity with the enzyme. Guinea pig anti-rat liver catalase antibody and rabbit anti-rat liver catalase antibody were prepared as described previously (
Protein AGold and IgGGold Probes
Two kinds of colloidal gold (15 nm and 8.5 nm in diameter) were prepared by the method of
Differential Centrifugation
Ten percent rat liver homogenate (W/V) in 0.2 M mannitol + 10 mM HEPESKOH (pH 7.4) + 0.2% ethanol was prepared in a PotterElvehjem homogenizer with a Teflon pestle. The homogenate was centrifuged at 1900 x g for 10 min with a Kubota Centrifuge 1710 (Kubota; Tokyo, Japan) using an RA228J rotor to remove cell debris and nuclei. The supernatant was centrifuged at 22,000 x g for 20 min. The resulting pellet was gently suspended in homogenization solution and used as light mitochondrial fraction. The supernatant was centrifuged at 100,000 x g for 60 min with a Hitachi ultracentrifuge (Hitachi; Tokyo, Japan) using an RP50 rotor. The resulting pellet was referred to as the microsomal fraction and the supernatant the cytosolic fraction. Each fraction was stored at -70C until use.
Nycodenz Gradient Centrifugation
The method used was based on that described by
Enzyme Assay
Catalase, D-amino acid oxidase, and urate oxidase were assayed as peroxisomal marker enzymes. Catalase activity was measured as described by -hydroxyacid oxidase, and urate oxidase activities were assayed by a modification of the method reported for acyl-CoA oxidase activity (
Immunotitration
Quantitative precipitation reactions were carried out with the purified ICD2 and a fixed amount of the antibody in the presence of 0.15 M NaCl and 50 mM potassium phosphate, pH 7.5. After overnight incubation at 4C, the mixture was centrifuged at 10,000 x g for 20 min and an aliquot of the supernatant was used for enzyme assay. The precipitate was washed once with PBS, dissolved in 10 mM NaOH, and protein concentration was assayed.
Immunoblotting Analysis
Rat liver homogenate was prepared as described above. The fractions isolated by Nycodenz gradient centrifugation were diluted 10-fold with 20 mM HEPESKOH buffer, pH 7.4. These samples were mixed with the same volume of sample buffer of SDS-PAGE. The mixture was heated in boiling water for 2 min. 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 reaction.
Other Analytical Procedures
SDS-PAGE was carried out according to
Immunocytochemical Procedures
Immunofluorescence Microscopy.
Rat liver was fixed by perfusion with fixative for 10 min at RT. Fixative consisted of 4% paraformaldehyde, 0.2% glutaraldehyde, and 0.2 M HEPES-KOH buffer (pH 7.4). Small tissue blocks of fixed liver were incubated overnight in antifreeze solution consisting of 20% polyvinylpyrolidone (MW 10,000), 1.8 M sucrose, and 10 mM HEPESKOH (pH 7.4) at 4C (
Immunoelectron Microscopy Using Thin Cryosections.
Tissue samples used were the same as employed in immunofluorescence microscopy. Thin cryosections were cut with the same instrument according to the method of
Double-labeling by Postembedding Immunoelectron Microscopy. Fixation of rat liver was performed as described above. Tissue slices of fixed rat liver were cut into small blocks, which were then dehydrated in graded ethanol and embedded in LR White (London Resin; Reading, UK) at -20C. Polymerization of resin was performed overnight under UV light at -20C. Thin sections were cut with a diamond knife using a Leichert Ultracut R. The sections were double-labeled for ICD2 and catalase, using gold probes of two different sizes, and stained with uranyl acetate and lead citrate. All thin sections were examined with a Hitachi H7500 electron microscope.
Quantitative Analysis of Gold Labeling. Thin sections were obtained three tissue blocks and incubated with anti-ICD2 or IgG fraction from pre-immune serum (immunocytochemical control), followed by protein Agold. Ten micrographs were taken from the sections stained for ICD2 at a magnification of 15,000 and enlarged to positive pictures at a final magnification of x30,000. Similarly, 10 micrographs were taken from the control sections. A total of 60 micrographs, each of which contained a part of a hepatocyte, were quantitatively analyzed as follows. Areas of peroxisomes and cytoplasm, including ER, were estimated using a digitizer (Sigma Scan; Jandel Scientific, San Rafael, CA) attached to a computer. Gold particles on the peroxisomes and the cytoplasm were counted and labeling density in each compartment was calculated. The labeling density was expressed as gold particles per µm2 of the compartment. In addition, we estimated sharing of gold particles by each compartment in an area of 100 µm2 using the same micrographs that were used for measurement of the labeling density. Sharing was expressed as the percentage of gold particles on the compartments to total gold particles. The labeling density and the sharing were corrected by the corresponding values obtained similarly from the immunocytochemical control sections.
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Results |
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Purification of ICD2 and Specificity of Antibody to ICD2
ICD2 was purified 528-fold from 100,000 x g supernatant fraction of rat liver. The recovery was 33% and purified enzyme had a specific activity of 50.1 U/mg. The apparent molecular mass of the native enzyme was determined to be 47 kD by SDS-PAGE (Fig 1A). Antibody to purified ICD2 developed a single band on immunoblotting analysis, which migrated at a position corresponding to 47 kD (Fig 1B), indicating the monospecificity of the antibody.
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Immunotitration
The titration of the purified preparation of ICD2 by its antibody is shown in Fig 2. The antibody reacted with whole ICD2 added within a range of approximately 40 U added and left no enzyme activity of ICD2 in the supernatant after centrifugation. In the presence of an excess amount of antigen, the anti-ICD2 antibody precipitated approximately half of the antigen. These results indicated that our antibody to ICD2 could immunochemically react with ICD2.
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ICD Activity Distribution in the Subcellular Fractions Isolated by Differential Centrifugation
The results are shown in Fig 3. No urate oxidase activity was detected in the cytosolic fraction, with all activity present in the mitochondrial fraction. In the rat liver, this enzyme is contained in the crystalloid core of peroxisomes, which is insoluble at neutral pH. This was the reason why urate oxidase was detected only in the particulate fraction. In contrast, 65% to 50% of D-amino acid oxidase, catalase, and L--hydroxyacid oxidase activities were present in the mitochondrial fraction, whereas only 25% of ICD2 was found in the particulate fraction and 90% of the activity was present in the cytosolic fraction. Two oxidases other than urate oxidase and catalase appeared in the cytosolic fraction in different amounts.
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Isolation of Peroxisomes by Nycodenz Density Gradient Centrifugation of Rat Liver
The results are shown in Fig 4. The separation of peroxisomes from mitochondria and lysosomes was determined by the distribution of marker enzymes on the gradient. The intact peroxisomes, as detected by assay of catalase activity, were separated from the mitochondrial fraction and were located at higher density (left in Fig 4). The mitochondria, as measured by succinate cytochrome C reductase, the lysosomes, as determined by acid phosphatase, and the ER, as measured by esterase, were located at lower density (right in Fig 4). Significant ICD2 activity was detected in fractions 2124, which contained the cytosolic proteins and proteins released from ruptured peroxisomes or other organelles. Fractions 27 (peak of peroxisomal fractions) contained the majority of ICD2 activity (64.3%), whereas lower-density fractions 2024 included half of this ICD2 activity (32%).
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Immunochemical Distribution of ICD2 and Organelle Marker Enzymes in Nycodenz Gradients
We analyzed 24 fractions isolated on a Nycodenz gradient by immunoblotting. The results are shown in Fig 5. Strong signals for ICD2 were distributed bimodally in fractions 25 and 1824, where the activity was detected. Signals in the latter fractions were weaker than those in the former. The distribution pattern of catalase signal was quite similar to that of ICD2. Signals for acyl-CoA dehydrogenase, a mitochondrial marker, were present in fractions 2024 but not in higher-density fractions (fractions 25), where catalase and ICD2 were found.
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Double Immunofluorescence Staining of ICD2 and Catalase in Rat Liver
The results are shown in Fig 6. Green fluorescence showing ICD2 antigenic sites was localized in the cytoplasmic granules but not in other parts of the hepatocytes. All of the positive granules in the same sections were also stained for catalase (red color), indicating that ICD2-positive granules were peroxisomes.
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Immunoelectron Microscopic Localization of ICD2 in Ultracryosections of Rat Liver
Gold particles showing the antigenic sites of ICD2 were observed mainly in peroxisomes, but labeling over the cytoplasmic matrix was never above background levels (Fig 7). In peroxisomes, gold particles were scattered in the matrix surrounding the nucleoid core but were not concentrated in specific areas of the matrix. On immunocytochemical control sections incubated with pre-immune serum, no gold particles were noted on the peroxisomes or other compartments.
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Double-labeling of ICD2 and Catalase by Postembedding Electron Microscopy
There were no differences in the labeling intensity for ICD2 between cryosections and LR White sections. Thin sections of LR White-embedded rat liver were labeled for ICD2 and catalase by the double-labeling technique. Large gold particles (15 nm) showing ICD2 antigenic sites were observed mainly in peroxisomes (Fig 8). The same peroxisomes were also stained with small gold particles (8.5 nm) for catalase, clearly demonstrating that ICD2 and catalase were co-localized in the peroxisomes. No labeling for either antigen was noted in the cytoplasmic matrix.
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Quantitative Analysis of Immunolabeling for ICD2
The results are shown in Table 1. Labeling density in peroxisomes was significantly higher than in the cytosolic compartment. When we subtracted background labeling from the labeling density of each compartment, the peroxisomal labeling density was 1354-fold higher than the cytosol. Approximately 99.8% of gold labeling in areas of 100 µm2 was present in peroxisomes and 0.2% was found in the cytosol. In a textbook (
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Discussion |
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Purity of ICD2 Preparation and Anti-ICD2 Antibody
Specific activity of purified ICD2 was 528-fold higher than that of the starting material, which was similar to the values reported previously (
Distribution of ICD2 in Rat Liver
After differential centrifugation of rat liver homogenate, most of the ICD2 activity (75%) was found in the cytosolic fraction and only 25% of the activity was in the mitochondrial fraction containing peroxisomes. These results strongly suggest that ICD2 is a cytosolic enzyme. The other peroxisomal enzymes (catalase, D-amino acid oxidase, and L--hydroxyacid oxidase), with the exception of urate oxidase, were also detected in the cytosolic fraction in various amounts. All these enzymes have been shown to be localized exclusively in the peroxisomes of the liver and kidney by immunoelectron microscopy (
-hydroxyacid oxidase are associated with the central clear matrix and the marginal plate of peroxisomes, respectively (
Subcellular Localization of ICD2 in Rat Hepatocytes
The enzyme activity of ICD2 was detected in the peroxisomal fractions isolated by Nycodenz gradient centrifugation. Immunoblotting analysis of the same fractions showed that ICD2 was co-localized with catalase but not with acyl-CoA dehydrogenase (a mitochondrial marker). ICD2 was also detected in lower-density fractions in which catalase, succinate cytochrome C reductase, acid phosphatase (a lysosomal marker), and esterase (an ER marker) co-existed. Catalase activity in the fractions may be derived from peroxisomes aggregated with the other organelles and the enzyme released from peroxisomes. These results demonstrated that ICD2 is associated with peroxisomes. Double immunofluorescence staining of ICD2 and catalase showed that these two enzymes were co-localized mainly in peroxisomes. If the enzyme were localized in the cytosol, its levels would be very low. Moreover, immunoelectron microscopic localization of ICD2 determined by two different methods also demonstrated that ICD2 is contained mainly in the peroxisomes. Double-labeling experiments showed that ICD2 and catalase were co-localized in the same peroxisomes but mostly not in the other compartments including the cytoplasmic matrix. Quantitative analysis of the results of postembedding immunoelectron microscopy revealed that ICD2 was localized predominantly in the peroxisomes. Because labeling density mostly reflects the concentration of the antigens in a compartment, our results showed that liver peroxisomes contain ICD2 at levels 1354-fold higher than the cytosol. Moreover, 99.8% and 0.2% of total gold particles found in 100-µm2 areas of tissue sections were present in the peroxisomes and the cytoplasm, respectively. According to a textbook (
Isozymes of ICD2 have been found in various tissues (
Function of ICD2 in Peroxisomes
In mammals and yeast, three isozymes of ICD are known: mitochondrial NAD-specific ICD, NADP-specific ICD1 (mitochondria type), and ICD2 (cytosolic type). NAD-specific ICD catalyzes a key step in the tricarboxylic acid cycle, whereas the physiological roles of two NADP-specific enzymes are not clearly understood. It is well known that peroxisomes break down very long-chain fatty acids and unsaturated fatty acids by ß-oxidation. In the ß-oxidation of unsaturated fatty acids, NADPH-dependent 2,4-dienoyl-CoA reductases and 3-cis-
2-trans-enoyl-CoA isomerases are required to remove the double bonds (
2,
4-dienoyl-CoA reductase in ß-oxidation of unsaturated fatty acids with double bonds at even-numbered carbons. It has been shown that NADPH required in the reaction of NADPH-dependent 2,4-dienoyl-CoA reductases in yeast is provided by peroxisomal ICD (
Received for publication December 8, 2000; accepted April 11, 2001.
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