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.     

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD⁺ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.     

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.     

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.     

Peroxisomal targeting signals in green algae

Peroxisomal enzymatic proteins contain targeting signals (PTS) to enable their import into peroxisomes. These targeting signals have been identified as PTS1 and PTS2 in mammalian, yeast, and higher plant cells; however, no PTS2-like amino acid sequences have been observed in enzymes from the genome database of Cyanidiochyzon merolae (Bangiophyceae), a primitive red algae. In studies on the evolution of PTS, it is important to know when their sequences came to be the peroxisomal targeting signals for all living organisms. To this end, we identified a number of genes in the genome database of the green algae Chlamydomonas reinhardtii, which contains amino acid sequences similar to those found in plant PTS. In order to determine whether these sequences function as PTS in green algae, we expressed modified green fluorescent proteins (GFP) fused to these putative PTS peptides under the cauliflower mosaic virus 35S promoter. To confirm whether granular structures containing GFP–PTS fusion proteins accumulated in the peroxisomes of Closterium ehrenbergii, we observed these cells after the peroxisomes were stained with 3, 3′-diaminobenzidine. Our results confirm that the GFP–PTS fusion proteins indeed accumulated in the peroxisomes of these green algae. These findings suggest that the peroxisomal transport system for PTS1 and PTS2 is conserved in green algal cells and that our fusion proteins can be used to visualize peroxisomes in live cells.     

Activated oxygen‐mediated metabolic functions of leaf peroxisomes

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. The presence in these organelles of superoxide dismutases and the generation of superoxide radicals (O₂^??) was first demonstrated in plant tissues and in recent years different experimental evidence has suggested the existence of cellular functions related to activated oxygen species. Some of these functions are analyzed in this work. In purified intact peroxisomes from pea (Pisum sativum L.) leaves, xanthine oxidase and urate oxidase were found to be present. The occurrence and the level of the metabolites xanthine, hypoxanthine, uric acid, and allantoin were studied in extracts of pea leaf peroxisomes by HPLC. Xanthine, uric acid, and allantoin were detected in peroxisomes. These results suggest a cellular role for leaf peroxisomes in the catabolism of purines. In peroxisomal membranes, 3 polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa, respectively, have been shown to generate superoxide radicals. These PMPs were purified from pea leaf peroxisomal membranes and characterized. While the 18- and 32-kDa PMPs use NADH as electron donor for O₂^?? production, the 29-kDa PMP was clearly dependent on NADPH. Very recently, the occurrence in pea leaf peroxisomes of all the enzymes of the ascorbate-glutathione cycle has been demonstrated. NADPH is required for the glutathione reductase activity of the cycle and this implies the reduction of NADP⁺ to NADPH. This recycling function could be carried out by the NADP-dependent glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and isocitrate dehydrogenase (ICDH). These 3 dehydrogenases have been demonstrated to be present in the matrix of pea leaf peroxisomes. The catabolism of purines, the superoxide-generating PMPs, the ascorbate-glutathione cycle, and the dehydrogenase-mediated recycling of NADPH, are activated oxygen roles of leaf peroxisomes that add to other functions previously known for peroxisomes from eukaryotic cells.     

The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

We studied the chronological lifespan of glucose‐grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome‐deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β‐oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β‐oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β‐oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β‐oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions.     

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca²⁺-removal and towards the dimer upon Ca²⁺-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca²⁺/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.     

The plant PTS1 receptor: similarities and differences to its human and yeast counterparts

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon ( Citrullus vulgaris ) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X₂-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.     

异柠檬酸脱氢酶基因调控对拉格酵母抗自溶性能的影响

啤酒酵母的自溶会影响啤酒的风味和品质,对酵母自溶的调控是工业啤酒生产的迫切需求。前期在啤酒酵母自溶的研究中发现柠檬酸循环相关基因对酵母自溶有较大影响。为探究柠檬酸循环中异柠檬酸脱氢酶基因调控对自溶的影响,在典型拉格啤酒酵母(Saccharomyces pastorianus H.)Pilsner中对IDP1和IDP2基因进行了破坏和过表达。结果发现IDP1基因的破坏能提高酵母的抗自溶能力,自溶96 h抗自溶指数为8.40,比原始菌提高了1.5倍。IDP1基因的破坏提高还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)的供应,NADPH/NADP^(+)的比值为1.94,发酵结束时胞内ATP水平比原始菌提高1.8倍,活性氧(reactive oxygen species,ROS)相比原始菌减少10%。IDP2基因的缺失造成酵母快速自溶和NADPH供应的减少,自溶96 h抗自溶指数为4.03,NADPH/NADP^(+)的比值为0.89。发酵结束时胞内ATP水平相比原始菌减少8%,ROS是原始菌的1.3倍。本研究进一步阐释了柠檬酸循环相关基因对酵母自溶的调控机理,为选育自溶性能可控的优良酵母提供了理论基础。     

Antioxidant cytoprotection by peroxisomal peroxiredoxin-5

Peroxiredoxin-5 (PRDX5) is a thioredoxin peroxidase that reduces hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This enzyme is present in the cytosol, mitochondria, peroxisomes, and nucleus in human cells. Antioxidant cytoprotective functions have been previously documented for cytosolic, mitochondrial, and nuclear mammalian PRDX5. However, the exact function of PRDX5 in peroxisomes is still not clear. The aim of this work was to determine the function of peroxisomal PRDX5 in mammalian cells and, more specifically, in glial cells. To study the role of PRDX5 in peroxisomes, the endogenous expression of PRDX5 in murine oligodendrocyte 158 N cells was silenced by RNA interference. In addition, human PRDX5 was also overexpressed in peroxisomes using a vector coding for human PRDX5, whose unconventional peroxisomal targeting sequence 1 (PTS1; SQL) was replaced by the prototypical PTS1 SKL. Stable 158 N clones were obtained. The antioxidant cytoprotective function of peroxisomal PRDX5 against peroxisomal and mitochondrial KillerRed-mediated reactive oxygen species production as well as H₂O₂ was examined using MTT viability assays, roGFP2, and C11-BOBIPY probes. Altogether our results show that peroxisomal PRDX5 protects 158 N oligodendrocytes against peroxisomal and mitochondrial KillerRed- and H₂O₂-induced oxidative stress.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.     

The human PICD gene encodes a cytoplasmic and peroxisomal NADP(+)-dependent isocitrate dehydrogenase.

Human PICD was identified by homology probing the data base of expressed sequence tags with the protein sequence of Saccharomyces cerevisiae Idp3p, a peroxisomal NADP(+)-dependent isocitrate dehydrogenase. The human PICD cDNA contains a 1242-base pair open reading frame, and its deduced protein sequence is 59% identical to yeast Idp3p. Expression of PICD partially rescued the fatty acid growth defect of the yeast idp3 deletion mutant suggesting that PICD is functionally homologous to Idp3p. Kinetic studies on bacterially expressed PICD demonstrated that this enzyme catalyzed the oxidative decarboxylation of isocitrate to 2-oxoglutarate with a specific activity of 22.5 units/mg and that PICD displayed K(M) values of 76 microM for isocitrate and 112 microM for NADP(+). In subcellular fractionation experiments, we found PICD in both peroxisomes and cytoplasm of human and rat liver cells, with approximately 27% of total PICD protein associated with peroxisomes. The presence of PICD in mammalian peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. As for cytoplasmic PICD, the phenotypes of patients with glucose-6-phosphate dehydrogenase deficiency (Luzzatto, L., and Mehta, A. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) Vol. 3, 7th Ed., pp. 3367-3398, McGraw-Hill Inc., New York) suggest that PICD serves a significant role in cytoplasmic NADPH production, particularly under conditions that do not favor the use of the hexose monophosphate shunt (Luzzatto et al.).     

Regulation of peroxisome size and number by fatty acid beta -oxidation in the yeast yarrowia lipolytica

The Yarrowia lipolytica MFE2 gene encodes peroxisomal beta-oxidation multifunctional enzyme type 2 (MFE2). MFE2 is peroxisomal in a wild-type strain but is cytosolic in a strain lacking the peroxisomal targeting signal-1 (PTS1) receptor. MFE2 has a PTS1, Ala-Lys-Leu, that is essential for targeting to peroxisomes. MFE2 lacking a PTS1 can apparently oligomerize with full-length MFE2 to enable targetting to peroxisomes. Peroxisomes of an oleic acid-induced MFE2 deletion strain, mfe2-KO, are larger and more abundant than those of the wild-type strain. Under growth conditions not requiring peroxisomes, peroxisomes of mfe2-KO are larger but less abundant than those of the wild-type strain, suggesting a role for MFE2 in the regulation of peroxisome size and number. A nonfunctional version of MFE2 did not restore normal peroxisome morphology to mfe2-KO cells, indicating that their phenotype is not due to the absence of MFE2. mfe2-KO cells contain higher amounts of beta-oxidation enzymes than do wild-type cells. We also show that increasing the level of the beta-oxidation enzyme thiolase results in enlarged peroxisomes. Our results implicate peroxisomal beta-oxidation in the control of peroxisome size and number in yeast.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism.     

Synthesis of glycolytic and peroxisomal enzymes in Tetrahymena following a change in culture conditions

The specific activity of a peroxisomal enzyme, lactate oxidase, and of pyruvate kinase and lactate dehydrogenase, which are not peroxisomal, increased rapidly when shaken cultures of Tetrahymena were transferred to conditions of oxygen restriction and supplemented with glucose. Two other peroxisomal enzymes, catalase and TPN-linked isocitrate dehydrogenase, did not increase substantially, nor did succinate dehydrogenase. The increases were reduced if glucose was not added at the time of transfer, and were prevented by actinomycin D or cycloheximide, but not by chloramphenicol. The results suggest an involvement of peroxisomes in the metabolism of glycolytic endproducts when the availability of oxygen to the cell is limiting.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.