Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

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 49:1123-1131, 2001)     

Cellular localization of α-ketoglutarate: Glyoxylate carboligase in rat tissues

α-Ketoglutarate : glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the α-ketoglutarate decarboxylase moiety of the α-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of α-ketoglutarate decarboxylase and of α-ketoglutarate dehydrogenase complex.The data indicate that α-ketoglutarate : gloxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from another laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.     

Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other mitochondrial enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.     

Studies on mouse liver urate oxidase

Summary Distribution of urate oxidase in subcellular components such as nuclei, mitochondria, lysosomes, microsomes, and cell sap, was investigated by both enzymatic and immunochemical methods. The subcellular components were prepared from mouse liver homogenate by differential centrifugation and the resulting microbody-rich mitochondrial fraction was fractionated by sucrose density gradient centrifugation. The enzymatically determined urate oxidase was distributed mainly in mitochondrial and lysosome fractions. The immunochemically assayed urate oxidase antigen was localized in mitochondrial, lysosome, and microsome fractions. The antigen to enzyme ratio was 1.0 in the mitochondrial and lysosome fractions, and about 2.0 in the microsome fraction.Sucrose density gradient centrifugation of the mitochondrial fraction indicated that the urate oxidase antigen was distributed around three density bands of 1.07, 1.15, and 1.24. The main band (1.24) was consistent with the microbody fraction. From these results, it was suggested that a precursor protein (proenzyme) might be located in the microsome fraction.This work was supported in part by a grant 777007 from the Ministry of Education, Japan, in 1972.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Alanine:glyoxylate aminotransferase is present as the apoenzyme in the peroxisomes of chicken kidney

The subcellular distribution of alanine:glyoxylate aminotransferase in chicken kidney was examined by centrifugation in a sucrose density gradient. The enzyme was found to be present as the apoform in the peroxisomes and as the holoform in the mitochondria. Alanine:glyoxylate aminotransferase in different mammalian kidneys were all present as the holoenzyme in the mitochondrial and soluble fractions.     

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.     

Subcellular localization of renin and kallikrein in rat kidney.

The subcellular localization of renin and kallikrein in rat kidney cortex homogenate was investigated using both differential and density gradient centrifugation techniques. Highest specific activity of renin was found in the heavy mitochondrial fraction. Mitochondrial localization of renin was further supported by the behaviour of succinic dehydrogenase. By differential centrifugation, highest specific activity of kallikrein was found in the light mitochondrial fraction, while by density gradient centrifugation kallikrein was almost completely recovered in the lysosomal fraction. Lysosomal localization of kallikrein is further supported by the behaviour of acid phosphatase. The different subcellular localizations of renin and kallikrein are confirmed and the suggestion that kallikrein is located in the lysosomes is advanced.     

Study on chemical species of iodine in human liver

The distribution and chemical species of iodine in various subcellular fractions of human liver were studied by using epithermal neutron activation analysis combined with chemical and biochemical separation techniques, such as gradient centrifugation and gel chromatography. It was found that the total iodine content orders in various subcellular fractions is as follows: nuclei > cytosol > mitochondria > lysosome > microsome. In the lysosomal fraction, iodine is mainly bound to macromolecules, whereas in the nuclei and mitochondrial fractions, mainly with lower-molecular-weight organic compounds. In the cytosol fraction, iodine is combined with three proteins, in which iodine is chiefly bound with mid- and high-molecular-weight proteins.     

The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

Free Mitochondria and Synaptosomes from Single Rat Forebrain. A Comparison Between Two Known Subfractionation Techniques

Two published subcellular subfractionation techniques employing Ficoll-sucrose or sucrose-density gradient centrifugation, respectively, are evaluated for their capacity to yield fractions containing free mitochondria and synaptosomes from a single rat forebrain. The enzymes lactate dehydrogenase, acetylcholinesterase, NAD(P)H-cytochrome c reductase, and citrate synthase, markers of different subcellular components, were used to assess the purity and integrity of the fractions. Judged by the distribution of these specific enzymatic markers, the free mitochondria obtained by the Ficoll-sucrose gradient technique were less contaminated by synaptosomes and had greater biochemical integrity than those obtained by the sucrose-gradient technique. By contrast, the synaptosomes obtained by the Ficoll-sucrose gradient technique resulted in more contamination by microsomes than those prepared in a sucrose gradient.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.     

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.     

Intracellular localization of aldehyde dehydrogenase in rat liver

1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 mumol/min per g, with an apparent K(m) value below 10(-5)m. The highest rate of activity was observed in phosphate buffers of high P(i) concentration (above 60mm). 3. The activity measured was completely dependent on NAD(+). 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed.