Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis

Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis. Received: 11 March 1997 / Accepted: 24 June 1997     

Conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol into 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rabbit liver mitochondria

Rabbit liver mitochondria in the presence of NAD+ were found to catalyze the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol into 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid. The peroxisomal fraction did not catalyze the reaction. Sonication of the mitochondria or dialysis overnight against a hypotonic buffer increased the rate of oxidation twofold. Most of the enzyme activity was recovered in the supernatant fraction after centrifugation at 100,000xg of sonicated mitochondria. 4-Heptylpyrazole, an inhibitor of cytosolic ethanol dehydrogenase, inhibited the mitochondrial formation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by 70%. Disulfiram, an inhibitor of cytosolic acetaldehyde dehydrogenase, did not inhibit the reaction. The role of the mitochondrial dehydrogenase system in bile acid biosynthesis is discussed.     

Induction of a Peroxisomal Malate Dehydrogenase Isoform in Liver of Starved Rats

The influence of starvation on malate dehydrogenase (MDH) in rat liver was investigated. Native electrophoresis revealed two MDH isoforms in non-starved rats and three isoenzymes in starved rats. After sucrose density gradient centrifugation of cell organelles from liver, MDH activity was detected in the mitochondrial and cytosolic fractions from non-starved rats. However, additional activity was found in the peroxisomal fraction from starved rats. The latter was identified as the electrophoretically new isoform in starved animals. The three isoforms of malate dehydrogenase from hepatocytes were separated and partially purified by chromatography on DEAE-Toyopearl. Several kinetic and regulatory properties of the three isoforms were rather similar. It is suggested that the newly expressed isoform of MDH operates in the glyoxylate cycle of liver peroxisomes of food-starved animals.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

Subcellular Distribution of Human Brain Aldehyde Dehydrogenase

Abstract: Two human brain surgery biopsies and one autopsy sample were subjected to subcellular fractionation. With either 0.12 or 6 mM-acetaldehyde as substrate, about half of the total aldehyde dehydrogenase activity was found in the mitochondrial (+ synaptosomal) fraction and less activity in the cytosolic, nuclear, and microsomal fractions. High-affinity activity was found only in the mitochondrial fraction. The enzyme in all fractions had a higher affinity for indole-3-acetaldehyde than for acetaldehyde. The kinetic data indicate the presence of several distinct aldehyde dehydrogenase isozymes that have ample capacity to oxidize both aliphatic and aromatic aldehydes in human brain.     

Investigation of the presence of branched-chain alpha-keto acid dehydrogenase in mammalian hepatic peroxisomes.

1. Rat liver was fractionated into peroxisomes and mitochondria and branched-chain keto acid (BCKA) dehydrogenase activity was measured. 2. All BCKA dehydrogenase activity was associated with the mitochondrial fraction and none with the peroxisomal fraction. 3. BCKA dehydrogenase was also not detected in hepatic peroxisomes of rats treated with clofibrate which induces several peroxisomal enzymes. 4. Hepatic peroxisomes from rabbit, hamster and dog also did not show any BCKA dehydrogenase activity. 5. We conclude that mammalian hepatic peroxisomes do not contain BCKA dehydrogenase.     

Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development

The specific activities of the enzymes of the tricarboxylic acid cycle; citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were determined in early fifth-stage, young and mature adult Obeliscoides cuniculi, the rabbit stomach worm. ∝-Ketoglutarate dehydrogenase activity could not be determined in any fraction. Fumarate reductase activity was found only in the mitochondrial fraction while all other enzymes, including an NADP-dependent malic enzyme were localized in the cytoplasm. Glutamate dehydrogenase, acid and alkaline phosphatase activities were also recorded. High levels of those enzymes acting in the “reversed” direction, i.e. MDH and fumarase relative to the enzymes of the “forward” direction, i.e. citrate synthase, aconitase and isocitrate dehydrogenase suggests that under anaerobic conditions a modified tricarboxylic acid cycle can operate. Some variations in specific activities were apparent as the worms matured but no qualitative differences were observed.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

Localization of Some Enzymes of β-oxidation of Fatty Acids in the Peroxisomes of Tetrahymena*

Mitochondria and peroxisomes were prepared from homogenates of Tetrahymena pyriformis by sedimentation through sucrose gradients. Catalase and isocitrate lyase served as peroxisomal markers; lactic dehydrogenase and glutamic dehydrogenase as mitochondrial markers. Acetyl-CoA synthetase, octanoyl-CoA synthetase, palmitoyl-CoA synthetase, 3-β hydroxyacyl CoA dehydrogenase, and thiolase activities were found in both the peroxisomes and the mitochondria. It is suggested that β-oxidation of fatty acids accurs in both organelles in Tetrahymena.     

Subcellular and Perisynaptic Distribution of Rat Brain Aldehyde Dehydrogenase Activity

Abstract: The nuclear mitochondrial and synaptosomal fractions of rat brain were each found to contain some 25–30% of the total aldehyde dehydrogenase activity. The cytoplasmic fraction had a very low total aldehyde dehydrogenase activity. There were differences in the distribution of the activity when different aldehydes were used as substrates, suggesting the presence of isoenzymes in the various subcellular compartments. When rats were treated intra-cisternally with 6-hydroxydopamine there was no change in brain aldehyde dehydrogenase activity, although the noradrenaline content and the activities of tyrosine hydroxylase and dopamine-β-hydroxylase were markedly decreased. Treatment with 6-hydroxydopamine also had no significant effect on the aldehyde dehydrogenase activity in retinal homogenates. The results suggest that the aldehyde dehydrogenase activity in rat brain is predominantly outside the catecholaminergic nerve terminals.     

Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast

Isozymes of NADP⁺-specific isocitrate dehydrogenase (IDP) provide NADPH in cytosolic, mitochondrial, and peroxisomal compartments of eukaryotic cells. Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments. However, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value. Mouse IDP2 was found to similarly colocalize in both cellular compartments when expressed in yeast at levels equivalent to those of endogenous yeast isozymes. The mouse enzyme can compensate for loss of yeast cytosolic IDP2 and of peroxisomal IDP3. Removal of the peroxisomal targeting signal of the mouse enzyme precludes both localization in peroxisomes and compensation for loss of yeast IDP3.     

Localisation of β-oxidation enzymes in peroxisomes of rice coleoptiles

Enzymes of the β-oxidation pathway in rice ( Oryza sativa L., cv. Arborio) coleoptiles were investigated. The coleoptiles contain acyl-CoA oxidase (EC 1.3.99.3), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase (EC 4.2.1.17) and thiolase (EC 2.3.1.9). Analysis of coleoptile homogenates by sucrose density fractionation showed a preferential distribution of these enzymes in the unspecialized peroxisomes. The enzymatic activity found in the mitochondrial fraction was due to peroxisomal contamination since electron micrographs show the peroxisomes to be intact and pure whereas the mitochondrial fraction was contaminated by other organelles. It appears that the β-oxidation pathway is localized in the unspecialized peroxisomes of rice coleoptiles, extending the number of plant species in which such a localization has been observed.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

Cellular and Subcellular Localization of Hexokinase, Glutamate Dehydrogenase, and Alanine Aminotransferase in the Honeybee Drone Retina

Abstract: Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[³H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.     

Aldehyde dehydrogenase (ALDH) isozymes in the gray short-tailed opossum (Monodelphis domestica): Tissue and subcellular distribution and biochemical genetics of ALDH3

Polyacrylamide gel isoelectric focusing (PAGE-IEF), cellulose acetate electrophoresis, and histochemical techniques were used to examine the tissue and subcellular distribution, genetics and biochemical properties of aldehyde dehydrogenase (ALDH) isozymes in a didelphid marsupial, the gray short-tail opossum (Monodelphis domestica). At least 14 zones of activity were resolved by PAGE-IEF and divided into five isozyme groups and three ALDH classes, based upon comparisons with properties previously reported for human, baboon, rat, and mouse ALDHs. Opossum liver ALDHs were distributed among cytosol (ALDHs 1 and 5) and large granular (mitochondrial) fractions (ALDHs 2 and 5). Similarly, kidney ALDHs were distributed between the cytosol (ALDH5) and the mitochondrial fractions (ALDHs 2, 4, and 5), whereas a major isozyme (ALDH3), found in high activity in cornea, esophagus, ear pinna, tail, and stomach extracts, was localized predominantly in the cytosol fraction. Phenotypic variants of the latter enzyme were shown to be inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal, ear pinna, tail, and stomach ALDH3 and supported biochemical evidence from other mammalian species that this enzyme has a dimeric subunit structure.     

Glycerol phosphate dehydrogenase in mammalian peroxisomes

Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD⁺:α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min^?1 mg^?1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD⁺:α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD⁺:α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands.Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K_m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K_m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD⁺:α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.     

An improved procedure for the simultaneous purification in high yield of peroxisomes,lysosomes, and mitochondria from rat liver

Summary Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.     

Peroxisomal localization and function of NADP-specific isocitrate dehydrogenases in yeast

Yeast peroxisomal NADP⁺-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for β-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2^+CKL) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2^+CKL. To test a potential cellular limit on peroxisomal levels, authentic yeast IDP3, which is normally strictly peroxisomal, was over-expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.