NADP-dependent Enzyme Research Articles

Human renal 11β-hydroxysteroid dehydrogenase 1 functions and co-localizes with COX-2

The local renal metabolism of glucocorticoids (GCs) by isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) determines their biological effects. 11β-HSD2, located in collecting duct epithelial cells of the mammalian and human kidney, serves as a putative “guardian” preventing GCs from binding to mineralocorticoid receptors. Various investigators have shown that both isoforms are present in kidney tissue from the rat, dog and other mammals. There is controversy as to whether 11β-HSD1 exists and functions in human kidney. The current studies examine the locale and function of both isoforms in human kidney. The expression of 11β-HSD1 was similar to that of 11β-HSD2 by Western blot. Two distinct Lineweaver Burke plots could be drawn providing enzyme kinetics for both isoforms. The apparent Km for the NADP dependent 11β-HSD1 enzyme was 0.42 μM while the apparent Km for the NAD dependent 11β-HSD2 enzyme was 10.2 nM. Human renal 11β-HSD1 appears to function as a dehydrogenase with no significant “reverse” reductase activity. Using immuno-histochemistry and Western blot analysis, 11β-HSD1 was found to co-localize with COX-2 in proximal tubule cells; COX-2 was not seen with 11β-HSD2 in cortical collecting duct. Thus, normal human kidney contains active 11β-HSD1 and 11β-HSD2. 11β-HSD1 co-localizes with COX-2 in proximal tubule cells.

Changes in Enzymes Associated with Energy Metabolism during the Early Post Mortem Period in Longissimus Thoracis Bovine Muscle Analyzed by Proteomics

Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADP-dependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle.

Gallic acid and hydrolysable tannins are formed in birch leaves from an intermediate compound of the shikimate pathway

Gallic acid is the starting material for hydrolysable tannin synthesis, but the mechanism of its formation in higher plants has not been known. To elucidate the pathway of gallic acid synthesis in the leaves of mountain birch ( Betula pubescens ssp. czerepanovii), we studied the effects of glyphosate (N-[phosphonomethyl]-glycine) on the levels of individual hydrolysable tannins. Glyphosate is known to block 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme of the shikimate pathway, thus reducing the synthesis of aromatic amino acids and phenolics derived from phenylalanine. However, when birch leaves were treated with glyphosate (1 mM, 72 h), the contents of individual hydrolysable tannins increased about twofold compared to control leaves. Our findings indicate that the immediate precursor of gallic acid may be an intermediate compound of the shikimate pathway before EPSP, most probably 3-dehydroshikimic acid. Consistently with this assumption, a highly active enzyme dehydroshikimate dehydrogenase (DSDG), which catalyses the direct conversion of 3-dehydroshikimic acid into gallic acid, was found in birch leaves. This new enzyme was partially purified to study its properties. It was found that DSDG is an NADP-dependent enzyme with a pH optimum at about 10.0. The enzyme showed an affinity for NADP + approximately 60-fold (K m=0.008 mM) that for dehydroshikimate (K m=0.49 mM). The intracellular organisation of the shikimate pathway and the hydrolysable tannin pathway are discussed.

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency ( k cat/ K m, NADPH ) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k cat/ K m, NADPH 1000-fold but had little effect on k cat/ K m, NADH . The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.

The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46 905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

Probing the Affinity and Specificity of Yeast Alcohol Dehydrogenase I for Coenzymes

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding βαβ fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178–Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2′-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants.

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway.

In this study we tested the hypothesis that the pentose phosphate pathway (PPP) participates in the meiotic induction of mouse oocytes. The electron acceptors methylene blue, phenazine ethosulfate (PES), and pyrroline-5-carboxylate (P5C) oxidize NADPH to NADP and activate the NADP-dependent enzymes of the PPP. Each of these compounds triggered a dose-dependent increase in meiotic maturation in hypoxanthine-arrested cumulus cell-enclosed oocytes during 17- to 18-h cultures. More than 96% of the oocytes underwent germinal vesicle breakdown (GVB) at the highest concentrations of P5C and PES tested (250 and 1 microM, respectively) as compared to only 45-52% of control oocytes. P5C was also stimulatory to denuded oocytes. Analysis of energy substrates in microdrop cultures revealed a 3.6-fold increase in glucose consumption by PES-treated oocyte-cumulus cell complexes that was associated with stimulation of GVB. On the other hand, 2-deoxyglucose, which interferes with glucose utilization, prevented the induction of maturation brought about by P5C. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase, prevented meiotic maturation in the presence or absence of FSH. Gonadotropin-induced maturation was also prevented by 6-aminonicotinamide (6-AN) and dehydroepiandrosterone (DHEA), inhibitors of the two NADP-dependent enzymes of the PPP, and this was accompanied by suppression of glucose consumption. Phosphoribosyl-pyrophosphate (PRPP) is an important compound required in purine metabolism and can be formed from the end product of the oxidative arm of the PPP, ribose-5-phosphate. Ribose, which can be metabolized to PRPP, increased PRPP synthesis in complexes and induced meiotic maturation when added to hypoxanthine-arrested cumulus cell-enclosed oocytes in glucose-free medium in both the presence and absence of FSH. PRPP levels within complexes were also increased by glucose and FSH, but were reduced by hypoxanthine, 6-AN, and DHEA. In addition, exogenous PRPP stimulated maturation in hypoxanthine-arrested oocytes. These results support the proposition that glucose metabolism through the PPP is important in the meiotic induction mechanism and may involve the generation of PRPP that acts, at least in part, through the purine metabolizing pathways.

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.

NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is targeted to the cytoplasm in insect cell lines

Cytosolic NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase synthetase and the mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) are differentially expressed during insect development although both enzymes are detectable at all stages. In contrast, cell lines derived from a variety of insect species express high levels of NMDMC but undetectable levels of the NADP-dependent enzyme. Northern analysis indicates the NMDMC message is expressed at levels 50–100 times higher in a Drosophila cell line compared to adult flies. RNase protection showed the predominance of shortened transcripts that require initiation at a downstream AUG producing a truncated protein that lacks a mitochondrial targeting sequence. These changes in expression effectively exchange the cytosolic NADP-dependent dehydrogenase for one with NAD specificity.

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.

Changes in the levels of 11 beta-hydroxysteroid dehydrogenase mRNA over the oestrous cycle in the rat.

The NADP dependent enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) metabolizes glucocorticoids to their inactive 11-keto-metabolites in a wide range of tissues. To date very little is known about the regulation of this enzyme at the level of gene transcription. In this study we show significant changes in the uterine, renal, ovarian and hepatic levels of 11 beta HSD1 mRNA over the oestrous cycle. Uterine and renal message levels followed the same pattern, with the highest levels observed at dioestrus and the lowest levels at oestrus, a pattern that correlates with plasma oestrogen levels during the cycle. In both the uterus and kidney 11 beta HSD1 message levels more than halved from dioestrus to oestrus, while renal levels than doubled at metoestrus. In contrast, hepatic 11 beta HSD1 message levels at prooestrus were twice those observed at metoestrus. Ovarian levels remain constant until metoestrus when a marked decrease in message levels was seen. 11 beta HSD1 mRNA levels are thus differentially regulated in a tissue specific manner throughout the oestrous cycle.

Cloning of the 11 beta HSD type II enzyme from human kidney.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to receptor inactive metabolites. Two isoforms of the enzyme exist. 11 beta HSD1 is a low affinity NADP dependent enzyme, while 11 beta HSD2 is a high affinity NAD dependent species thought to be responsible for endowing specificity on the mineralocorticoid receptor and for protecting the fetus from high circulating levels of maternal glucocorticoids. We have recently cloned the human renal 11 beta HSD2 enzyme. In this report we show that 11 beta HSD2 potently inactivates the synthetic glucocorticoid dexamethasone, producing a single product thought to be the 11-dehydrodexamethasone metabolite. Sequence analysis shows that the new isoform is a member of the short-chain alcohol dehydrogenase superfamily (SCAD), most closely related to 17 beta HSD2 and distantly related to 11 beta HSD1.

Evidence for distinct isoforms of 11β-hydroxysteroid dehydrogenase in the ovine liver and kidney

We have previously identified a unique 11β-hydroxysteroid dehydrogenase (11β-HSD) transcript in the ovine kidney. To examine whether this is indicative of a distinct isoform with respect to enzymatic activity, we studied and compared the characteristics of 11β-HSD activity in the ovine liver and kidney. 11β-HSD activity was determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. Although in both liver and kidney, the enzyme was localized by subcellular fractionation in the microsomes, the renal 11β-HSD displayed distinct characteristics in that it expressed only dehydrogenase activity and utilized almost exclusively NAD as cofactor (the respective activity in the presence of NAD and NADP was 190 ± 26 and 12 ± 2 pmol/min/mg protein). By contrast, the liver enzyme contained both dehydrogenase and reductase activities, and displayed preference for NADP and NADPH, respectively. Furthermore, with cortisol as substrate, the kidney 11β-HSD had a K m of 68 ± 7 nM which was over 100 times lower than the hepatic enzyme (8 ± 1 μM). In addition, the renal 11β-HSD activity was inhibited in a dose-dependent fashion by both carbenoxolone, a potent inhibitor of 11β-HSD, and the end product cortisone, whereas the liver enzyme showed little inhibition by either substance. In summary, these results provide strong evidence for the existence of distinct isoforms of 11β-HSD with respect to enzymatic activity in the ovine liver and kidney. In addition, the characteristics of the kidney enzyme closely resemble those of that described previously in the rabbit renal aldosterone target cells, and thus further demonstrating the presence of an isoform of 11β-HSD distinct from the NADP-dependent enzyme purified and cloned from the rat liver.

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.

Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family

Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.

Aldehyde dehydrogenases and their role in carcinogenesis.

Aldehydes are highly reactive molecules that may have a variety of effects on biological systems. They can be generated from a virtually limitless number of endogenous and exogenous sources. Although some aldehyde-mediated effects such as vision are beneficial, many effects are deleterious, including cytotoxicity, mutagenicity, and carcinogenicity. A variety of enzymes have evolved to metabolize aldehydes to less reactive forms. Among the most effective pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs). ALDHs are a family of NADP-dependent enzymes with common structural and functional features that catalyze the oxidation of a broad spectrum of aliphatic and aromatic aldehydes. Based on primary sequence analysis, three major classes of mammalian ALDHs--1, 2, and 3--have been identified. Classes 1 and 3 contain both constitutively expressed and inducible cytosolic forms. Class 2 consists of constitutive mitochondrial enzymes. Each class appears to oxidize a variety of substrates that may be derived either from endogenous sources such as amino acid, biogenic amine, or lipid metabolism or from exogenous sources, including aldehydes derived from xenobiotic metabolism. Changes in ALDH activity have been observed during experimental liver and urinary bladder carcinogenesis and in a number of human tumors, including some liver, colon, and mammary cancers. Changes in ALDH define at least one population of preneoplastic cells having a high probability of progressing to overt neoplasms. The most common change is the appearance of class 3 ALDH dehydrogenase activity in tumors arising in tissues that normally do not express this form. The changes in enzyme activity occur early in tumorigenesis and are the result of permanent changes in ALDH gene expression. This review discusses several aspects of ALDH expression during carcinogenesis. A brief introduction examines the variety of sources of aldehydes. This is followed by a discussion of the mammalian ALDHs. Because the ALDHs are a relatively understudied family of enzymes, this section presents what is currently known about the general structural and functional properties of the enzymes and the interrelationships of the various forms. The remainder of the review discusses various aspects of the ALDHs in relation to tumorigenesis. The expression of ALDH during experimental carcinogenesis and what is known about the molecular mechanisms underlying those changes are discussed. This is followed by an extended discussion of the potential roles for ALDH in tumorigenesis. The role of ALDH in the metabolism of cyclophosphamidelike chemotherapeutic agents is described. This work suggests that modulation of ALDH activity may an important determinant of the effectiveness of certain chemotherapeutic agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase, but not ferredoxin-NADP reductase, controls electron flow to phosphoglycerate in the dark-light transition

P700 is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P700 is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)(-1) electrons in geranium leaves, 16 nmol · (mg chlorophyll)(-1) in sunflower and 22 nmol · (mg chlorophyll)(-1) in oleander. Reduction of NADP during the initial phase of P700 oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.

Effect of thiabendazole and fenbendazole on glucose uptake and carbohydrate metabolism in trichuris globulosa

14C-glucose uptake by adult Trichuris globulosa was found to be a non-linear function of time and limiting substrate concentration. The uptake was a two component process, an initial rapid burst, followed by a lower steady state. Linear transformation of the substrate saturation kinetics by Eadie plot gave a K t value of 5×10 3 μM and J max of 5 μmol mg −1 dry weight min −1. Presence of the benzimidazole drugs, thiabendazole and fenbendazole, markedly inhibited the uptake process at concentrations in which the motility of the parasites was not affected. T. globulosa was found to possess the full complement of enzyme systems of glycolysis, Krebs cycle, phosphatases, transaminases and other NAD- and NADP-dependent enzymes. Thiabendazole and fenbendazole lowered the V max of most of these enzymes and affected the substrate-affinity constant (K m) in some of them. The mode of action of these drugs was discussed in the light of the data obtained.