NAD-dependent Enzyme Research Articles

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves

In higher plants formate dehydrogenase (FDH, EC 1.2.1.2.) is a mitochondrial, NAD-dependent enzyme. We previously reported that in potato (Solanum tuberosum L.) FDH expression is high in tubers but low in green leaves. Here we show that in isolated tuber mitochondria FDH is involved in formate-dependent O2 uptake coupled to ATP synthesis. The effects of various environmental and chemical factors on FDH expression in leaves were tested using the mitochondrial serine hydroxymethyltransferase as a control. The abundance of FDH transcripts is strongly increased under various stresses, whereas serine hydroxymethyltransferase transcripts decline. The application of formate to leaves strongly enhances FDH expression, suggesting that it might be the signal for FDH induction. Our experiments using glycolytic products suggest that glycolysis may play an important role in formate synthesis in leaves in the dark and during hypoxia, and in tubers. Of particular interest is the dramatic accumulation of FDH transcripts after spraying methanol on leaves, as this compound is known to increase the yields of C3 plants. In addition, although the steady-state levels of FDH transcript increase very quickly in response to stress, protein accumulation is much slower, but can eventually reach the same levels in leaves as in tubers.

NADP–Malic Enzyme from the C4PlantFlaveria bidentis:Nucleotide Substrate Specificity

NADP–malic enzyme (NADP–ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4dicotFlaveria bidentisand shown to possess intrinsic NAD-dependent malic enzyme activity. The NAD-dependent activity is optimal at pH 7.5 and in the presence of Mn2+. TheKmfor NAD is very high (20 mm), while theVmaxis 50% greater than theVmaxwith NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki∼2 μm). This very lowKireflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, theKmfor NADP is 1.5 μmwhile theKifor NADPH is 2 μm. Chicken liver NADP–ME also has NAD-dependent activity that is inhibited by low concentrations of NADPH. These results indicate that the NAD- and NADP-dependent activities are likely catalyzed by the same active site. The use of NAD as an alternative coenzyme revealed interactions between the binding of coenzyme and metal ions on theKmvalues of each of the other participants in the malic enzyme reaction. Thus, the affinity of malic enzyme for the divalent metal ions Mg2+and particularly Mn2+as well as the other substratel-malate is also dependent on the nucleotide coenzyme substrate. In turn, the divalent metal ion influences the affinity of the enzyme for the coenzyme as well asl-malate. With NADP as substrate theKmfor Mn2+is 4 μm, whereas with NAD theKmis 300 μm. The relatively high affinity of the enzyme for Mn2+and low affinity for NAD required the use of metal ion buffers when determining these values because of the substantial depletion of free Mn2+caused by binding to NADP.

Sorbitol Metabolism in Growing Tissues of Peach

Sorbitol is the major photosynthetic product in peach. In sink tissues, sorbitol is converted to fructose via the NAD-dependent enzyme sorbitol dehydrogenase (SDH). A new assay is described that allows rapid, simple quantitation of SDH activity in growing shoot tips, root tips, and fruits. The activity was measured on the crude extract desalted with a Saphadex G-25 column to eliminate small molecules such as sugars and nucleotides. Optimum buffer type and pH for the enzyme as well as degradation by proteolytic enzymes and stability over time were determined in the present study. Inhibition by dithiothreitol (DTT) was detected at an inhibitor concentration as low as 2 mM, proving the similarity with mammalian SDH. Storage of samples at 4°C overnight resulted in significant loss of enzyme activity. Using this assay, we also correlated SDH activity with sink strength in peach.

Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.

Tissue-Specific and Light-Mediated Expression of the C4 Photosynthetic NAD-Dependent Malic Enzyme of Amaranth Mitochondria.

In the C4 dicotyledonous grain plant amaranth (Amaranthus hypochondriacus), a mitochondrial NAD-dependent malic enzyme (NAD-ME; EC 1.1.1.39) serves a specialized and essential role in photosynthetic carbon fixation. In this study we have examined specialized photosynthetic gene expression patterns for the NAD-ME [alpha] subunit. We show here that the [alpha] subunit gene is preferentially expressed in leaves and cotyledons (the most photosynthetically active tissues), and this expression is specific to the bundle-sheath cells of these tissues from the earliest stages of development. Synthesis of the [alpha] subunit polypeptide and accumulation of its corresponding mRNA are strongly light-dependent, but this regulation is also influenced by seedling development. In addition, light-dependent accumulation of the [alpha] subunit mRNA is regulated at transcriptional as well as posttranscriptional levels. Our findings demonstrate that the NAD-ME of amaranth has acquired numerous complex tissue-specific and light-mediated regulation patterns that define its specialized function as a key enzyme in the C4 photosynthetic pathway.

Purification and partial characterization of bovine kidney aldehyde dehydrogenase able to oxidize retinal to retinoic acid.

A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1 and ALDH2. The enzymes ALDH1 and ALDH2 were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate--polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1 enzyme had a pI of 7.45 and exhibited a low Km (6.37 microM) for retinal, while the ALDH2 enzyme was found to have very low Km for acetaldehyde (0.98 microM). Based on its kinetic properties, it is suggested that the ALDH1 enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

Co-expression of two distinct isoforms of 11β-hydroxysteroid dehydrogenase in the ovine placenta

We have previously described two distinct isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) with respect to enzymatic activity in the ovine liver and kidney. To determine which isoform(s) is expressed in the ovine placenta, we studied the characteristics of 11 beta-HSD activity in placental tissues collected at days 140-143 of pregnancy. 11 beta-HSD activity was determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. At 100 nM cortisol, the placental 11 beta-HSD utilized NAD as cofactor, but displayed preference for NADP at 10 microM cortisol. Kinetic characteristics were examined in the presence of alternate cofactors, in order to determine whether this difference in the cofactor requirement represents distinct enzymes. With NAD as cofactor, the placental 11 beta-dehydrogenase had a Km (110 +/- 18 nM) compatible with the kidney enzyme, but displayed a Km (12 +/- 2 microM) similar/identical to the liver 11 beta-HSD when NADP was used. By contrast, the placental 11-oxoreductase showed preference for NADPH regardless of cortisone concentration. Kinetic analysis, using NADPH as cofactor, revealed a single species of 11-oxoreductase activity with a Km of 4 +/- 0.9 microM and a Vmax of 3.1 +/- 0.5 pmol/mg/min. Finally, since the NAD-dependent 11 beta-HSD in the ovine placenta displayed similar/identical kinetic characteristics to the enzyme described previously in the ovine kidney where a truncated 11 beta-HSD transcript was identified, we have also determined whether this transcript is expressed in the placenta by Northern blotting. It was found that the truncated 11 beta-HSD transcript was undetectable in the total RNA samples. These results demonstrate that both liver- and kidney-types of 11 beta-HSD activities are expressed in the ovine placenta, thus providing further evidence for the existence of a NAD-dependent 11 beta-HSD distinct from the well-characterized hepatic NADP-dependent enzyme. Furthermore, the lack of the truncated 11 beta-HSD transcript in the placenta suggests that the NAD-dependent enzyme identified in placenta and kidney is the product of a gene distinct from 11 beta-HSD.

Deoxyhypusine Synthase from Rat Testis: Purification and Characterization

Deoxyhypusine synthase is the first enzyme involved in the post-translational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formation of deoxyhypusine by transfer of the butylamine portion of spermidine to the epsilon-amino group of a specific lysine residue in the eIF-5A precursor. Its purification from rat testis was accomplished by ammonium sulfate fractionation and successive ion-exchange chromatographic steps, followed by chromatofocusing on a hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric focusing. Amino acid sequences of five tryptic peptides of the pure enzyme did not correspond to any sequences in the protein data banks. The enzyme migrates as a single band on SDS-polyacrylamide gel electrophoresis with an apparent monomer molecular mass of approximately 42,000 Da. Matrix-assisted laser desorption mass spectrometry gave a monomer mass of 40,800 Da. There is evidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitated deoxyhypusine synthase activity. The enzyme shows a strict specificity for NAD. Purified deoxyhypusine synthase catalyzes the overall synthesis of deoxyhypusine and, in the absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.

Crystal structure of a NAD-dependent d-glycerate dehydrogenase at 2·4 Å resolution

d-Glycerate dehydrogenase (GDH) catalyzes the NADH-linked reduction of hydroxypyruvate to d-glycerate. GDH is a member of a family of NAD-dependent dehydrogenases that is characterized by a specificity for the d-isomer of the hydroxyacid substrate. The crystal structure of the apoenzyme form of GDH from Hyphomicrobium methylovorum has been determined by the method of isomorphous replacement and refined at 2·4 Å resolution using a restrained least-squares method. The crystallographic R-factor is 19·4% for all 24,553 measured reflections between 10·0 and 2·4 Å resolution. The GDH molecule is a symmetrical dimer composed of subunits of molecular mass 38,000, and shares significant structural homology with another NAD-dependent enzyme, formate dehydrogenases The GDH subunit consists of two structurally similar domains that are approximately related to each other by 2-fold symmetry. The domains are separated by a deep cleft that forms the putative NAD and substrate binding sites. One of the domains has been identified as the NAD-binding domain based on its close structural similarity to the NAD-binding domains of other NAD-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. A model of a ternary complex of GDH has been built in which putative catalytic residues are identified based on sequence homology between the d-isomer specific dehydrogenases. A structural comparison between GDH and l-lactate dehydrogenase indicates a convergence of active site residues and geometries for these two enzymes. The reactionscatalyzed are chemically equivalent but of opposing stereospecificity. A hypothesis is presented to explain how the two enzymes may exploit the same coenzyme stereochemistry and a similar spatial arrangement of catalytic residues to carry out reactions that proceed to opposite enantiomers.

Isozymes of 11β-hydroxysteroid dehydrogenase: Which enzyme endows mineralocorticoid specificity?

Intracellular enzymes which interconvert circulating hormones between active and inactive forms aid in regulating the biological activity of the ligand in a cell-specific manner. This is particularly important in mineralocorticoid target tissues where glucocorticoids and mineralocorticoids have equivalent affinity for the mineralocorticoid receptor. Inactivation of glucocorticoids at the 11-hydroxyl position by the action of 11β-hydroxysteroid dehydrogenase (11β-OHSD) permits the occupation of the mineralocorticoid receptor by aldosterone in the presence of much higher levels of circulating cortisol. The suppression of dehydrogenase activity allows glucocorticoids to activate the mineralocorticoid receptor, leading to classical mineralocorticoid type effects such as sodium retention and potassium excretion. A number of 11β-OHSDs are currently candidate protectors of teh mineralocorticoid receptor. This review examines the attributes of these 11β-hydroxysteroid dehydrogenase isozymes and suggests reasons why a high affinity, NAD-dependent enzyme appears to be the most likely candidate to endow specificity on the mineralocorticoid receptor

Cloning and analysis of the C4 photosynthetic NAD-dependent malic enzyme of amaranth mitochondria.

In some C4 plant species, a mitochondrial NAD-dependent malic enzyme (EC 1.1.1.39) (NAD-ME) catalyzes the decarboxylation of 4 carbon malate in the bundle sheath cells, releasing CO2 for the Calvin cycle of photosynthesis. In amaranth, a dicotyledonous NAD-ME-type C4 plant, the photosynthetic NAD-ME purified as two subunits of 65 and 60 kDa, designated alpha and beta, respectively. Antiserum raised against the alpha subunit reacted only with the 65-kDa protein in immunoblot analysis. Immunogold electron microscopy using the alpha subunit antiserum demonstrated that this protein was localized specifically to the mitochondrial matrix of bundle sheath cells. The complete nucleotide sequence of a 2300-base pair alpha subunit cDNA clone showed that this gene encodes a protein that contains all of the motifs required for a complete and functional malic enzyme. The alpha subunit has significant similarity along its entire length to other known NAD- and NADP-dependent malic enzymes from plants, animals, and bacteria. The findings presented here provide new insights about the C4 photosynthetic NAD-ME and its evolutionary relationship to other forms of malic enzyme present in eukaryotic and prokaryotic organisms.

Metabolic alterations in cotyledons ofCucurbita pepoinfected by cucumber mosaic virus

Changes in the capacities of enzymes in various metabolic pathways have been measured during infection of cotyledons of Cucurbita pepo L. with cucumber mosaic virus (CMV). Starch accumulation and low sucrose content, which are characteristic of the early stages of infection, are reversed in the later stages of infection. The decline in starch correlated with a reduced capacity for starch synthesis (ADP-glucose pyrophosphorylase) and a rise in the capacity for starch degradation (total starch hydrolase, starch phosphorylase). 14CO2 feeding experiments, conducted at saturating C0 2 concentration, show that the newlyassimilated carbon was lost at a lower rate from infected cotyledons and less was incorporated into structural carbohydrates, phosphorylated intermediates plus organic acids, more into soluble sugars, amino acids and proteins. At a later stage of infection there were dramatic increases in respiratory capacity and a substantial alteration of carbohydrate metabolism. The infection had a large stimulatory effect on the capacity for oxidative pentose-phosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase), glycolysis (ATP- and pyrophosphate-dependent phosphofructokinases), tricarboxylic acid cycle (isocitrate dehydrogenase, fumarate hydratase), anaplerotic reactions (NADdependent malic enzyme, phosphoenolpyruvate carboxylase) and oxidative electron transport (cytochrome c oxidase). While there were no overall changes in photosynthetic rate (measured in saturating CO-J, infection either reduced (Rubisco and glycerate kinase) or did not affect (chloroplastic fructose bisphosphatase and hydroxypyruvate kinase) the capacities of the photosynthetic carbon reduction pathway or the photosynthetic carbon oxidation pathway.

Nitric oxide-independent, thiol-associated ADP-ribosylation inactivates aldehyde dehydrogenase.

Nitric oxide inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase and stimulates NAD-dependent automodification of a cysteine (Dimmeler, S., Lottspeich, F., and Brüne, B. (1992) J. Biol. Chem. 267, 16771-16774). Another NAD-utilizing dehydrogenase that has a catalytic cysteine, aldehyde dehydrogenase (ALDH), was also inhibited by nitric oxide. Unlike glyceraldehyde-3-phosphate dehydrogenase, ALDH was modified in a nitric oxide-independent process by ADP-ribose, but not by NAD. Modification, which proceeded to > 2 mol ADP-ribose.mol ALDH-1, was associated with an exponential decrease in enzyme activity to less than 10% of control. Two types of evidence suggested modification of the ALDH-active site: 1) ADP-ribose inhibited ALDH competitively (Ki = 0.46 mM) with respect to NAD (Km = 0.11 mM) in brief incubations and 2) the presence of substrates protected ALDH from both modification and inhibition by ADP-ribose. The ALDH-ADP-ribose bond was sensitive to base and mercuric ion and stable to acid and neutral hydroxylamine, properties shared with the ADP-ribosylcysteine linkage synthesized enzymatically by pertussis toxin. These data demonstrate a novel means of inactivation of an NAD-dependent enzyme, namely the affinity-based modification of the enzyme NAD site by ADP-ribose, and suggest that nonenzymatic ADP-ribosylation may be responsible for modification of cysteine residues.

Metabolism of acetaldehyde by rat isolated aortic rings: Does endothelial tissue contribute to its extrahepatic metabolism?

Acetaldehyde (AcH) metabolism in isolated aortic rings was studied by assessing in vitro-added AcH disappearing rate by head space gas chromatography. It was found that AcH was metabolized by aortic rings or by homogenates prepared in 0.1 M phosphate buffer containing Triton X-100, by an NAD-dependent enzyme with characteristics similar to those of aldehyde dehydrogenase (AIDH) present in mitochondria from rat liver and brain. This enzyme appears to be present in the vascular endothelium, since the action of aortic rings showed a remarkable decrease by its removal. Extrahepatic metabolism of AcH was assessed by the differences between AcH levels found in samples of blood obtained from the suprahepatic vein, carotid artery, femoral vein, and tail cut of rats. The in vitro activity of aortic rings, as well as the extrahepatic AcH metabolism, were significantly decreased by pretreatment of rats with disulfiram. The wide distribution of vascular endothelium throughout the body suggests that this tissue could contribute to AcH extrahepatic metabolism.

POSSIBLE ROLE OF MANNITOL 1-OXIDOREDUCTASE IN CELERY SINK TISSUE

Mannitol represents a major carbon storage compound in celery petioles. Recently, a novel mannitol dehydrogenase was isolated from celeriac (Apium graveoiens L. var. rapaceum) root tips and partially purified to a specific activity of 210 μmol/h/mg protein. This NAD-dependent enzyme catalyzes the oxidation of mannitol to mannose. not fructose, and this is uniquely different from the mannitol oxidation in lower organisms. Data are presented that show that the enzyme is also present in celery (Apium graveoiens L. var. dulce) sink tissue. The product of the mannitol oxidation in celery was determined to be mannose. Previous studies suggested that differences in fertilizer concentration in the root zone of celery could alter carbon partitioning between mannitol and hexose sugars and change enzyme activities. In this study, plants were grown hydroponically at two different salt concentrations, E.C.=2.7 mS/cm and E.C.=6.0 mS/cm. A high mannitol to hexose ratio was observed in celery petioles of plants grown at high salt concentration, supporting the hypothesis that the salt environment might alter both mannitol and hexose pools in a coordinate way. Plants grown at both salt concentrations did not differ in root or shoot fresh weight. These data support the hypothesis that mannitol might be involved in osmoprotection by acting as a compatible solute. Mannitol accumulation in celery petioles might be, in part, regulated through a mannitol catabolizing pathway involving mannitol oxidation to mannose by the NAD-dependent 1-oxidoreductase, mannose phosphorylation by mannose kinase, mannose-6-P conversion to fructose-6-P by phosphomannose isomerase and further conversion to hexoses through a sucrose intermediate. The novel mannitol 1-oxidoreductase might therefore be a key enzyme in controlling mannitol levels in sink tissues.

Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase.

Saccharomyces cerevisiae possess a monofunctional, cytoplasmic NAD-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase that converts 5,10-methylene-THF to 5,10-methenyl-THF (Barlowe, C. K., and Appling, D.R. (1990) Biochemistry 29, 7089-7094). We have now isolated the gene encoding this enzyme from a yeast genomic library using oligonucleotide probes based on internal peptide sequences from the purified protein. Nucleotide sequence analysis reveals a 320-amino acid open reading frame that contains both of the internal peptide sequences. The predicted molecular weight (36,236) is consistent with the estimated size (33,000-38,000) of the purified protein. Disruption of the chromosomal copy of the gene resulted in loss of NAD-dependent 5,10-methylene-THF dehydrogenase activity and led to a purine requirement in certain genetic backgrounds, confirming a role for this enzyme in the oxidation of cytoplasmic one-carbon units. A single gene was mapped to chromosome XI by hybridization to a yeast chromosomal blot. We propose MTD1 as the name for this gene. Northern analysis of total yeast RNA revealed a single transcript of approximately 1,100 nucleotides. Multiple transcription initiation sites were identified between 58 and 83 base pairs upstream of the start of translation. The amino acid sequences derived from the nucleic acid sequences of seven other methylene-THF dehydrogenases cloned to date have been found to be highly homologous. Although the predicted amino acid sequence of the yeast NAD-dependent enzyme shows slight homology to the other sequences, it appears to be only distantly related to the other 5,10-methylene-THF dehydrogenases.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO 2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3 1 21 or its enantiomer, with a = b = 131.2(7) A ̊ , c = 152.6(9) A ̊ , and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (λ = 0.95 Å) to about 3.0 Å resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

NADP-dependent 15-hydroxyprostaglandin dehydrogenase is homologous to NAD-dependent 15-hydroxyprostaglandin dehydrogenase and other short-chain alcohol dehydrogenases

Human tissues contain two enzymes that catalyze the oxidation of the 15-hydroxy group of prostaglandins: NAD-dependent 15-hydroxyprostaglandin dehydrogenase which is fairly specific for prostaglandins and NADP-dependent 15-hydroxyprostaglandin dehydrogenase with low specificity for prostaglandins. The recent determination of the amino acid sequence of the NAD-dependent enzyme has revealed similarities with a number of oxidoreductases with distinct specificities that constitute the short-chain alcohol dehydrogenase family. Comparison of the amino acid sequence of NADP-dependent 15-hydroxyprostaglandin dehydrogenase with the sequences of the NAD-dependent enzyme and other short-chain alcohol dehydrogenases revealed the same similarities, identifying the NADP-dependent enzyme as a short-chain alcohol dehydrogenase. The degree of homology between the two 15-hydroxyprostaglandin dehydrogenases is similar to the one between each of the two enzymes and other members of the family indicating an early evolutionary divergence of the two proteins.

Effects of pesticides on oxidative phosphorylation and electron transfer in potato tuber mitochondria

The monosodium salt of a synthetic auxin (2,4-D), and a quaternary ammonium salt (BrB), were tested on mitochondria isolated from fresh potato tubers. The two compounds inhibited mitochondrial respiration, the quaternary ammonium salt being the most effective. The uncoupling activity of 2,4-D resulted in the loss of energy production. Moreover, 2,4-D inhibited succinate dehydrogenase, malate dehydrogenase and NAD-dependent malic enzyme activities leading to a strong inhibition of electron flow. The mode of action of BrB was shown to be more complex. It was demonstrated that BrB acts on the membrane structure, its effects being concentration-dependent. Thus, low BrB concentrations inhibiting phosphorylation process by impeding phosphate entry to the matrix. In the presence of high BrB concentrations, mitochondrial membranes were permeable to cytochrome c. In addition, BrB efficiency diminished when the mitochondrial protein concentration was increased. Consequently, BrB probably caused, as a detergent, a large disorganization of the mitochondrial membranes.