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This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase ( DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed. [ABSTRACT FROM AUTHOR]

Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic conditions using a four-step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 ± 0.1 iron, 1.1 ± 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetaldehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N-terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a σ54-dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to σ54-dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression. [ABSTRACT FROM AUTHOR]

A cytosolic thyroid-hormone-binding protein (xCTBP), predominantly responsible for the major binding activity of T3 in the cytosol of Xenopus liver, has been shown to be identical to aldehyde dehydrogenase class 1 (ALDH1) [Yamauchi, K., Nakajima, J., Hayashi, H., Horiuchi, R. & Tata, J.R. (1999) J. Biol. Chem . 274 , 8460–8469]. Within this paper we surveyed which signaling, and other, compounds affect the thyroid hormone binding activity and aldehyde dehydrogenase activity of recombinant Xenopus ALDH1 (xCTBP/xALDH1) while examining the relationship between these two activities. NAD+ and NADH (each 200 µm), and two steroids (20 µm), inhibit significantly the T3 -binding activity, while NADH and NADPH (each 200 µm), and iodothyronines (1 µm), inhibit the ALDH activity. Scatchard analysis and kinetic studies of xCTBP/xALDH1 indicate that NAD+ and T3 are noncompetitive inhibitors of thyroid-hormone-binding and ALDH activities, respectively. These results indicate the formation of a ternary complex consisting of the protein, NAD+ and thyroid hormone. Although the in vitro studies indicate that NAD+ and NADH markedly decrease T3 -binding to xCTBP/xALDH1 at ≈ 10-4 m, a concentration equal to the NAD content in various Xenopus tissues, photoaffinity-labeling of [125 I]T3 using cultured Xenopus cells demonstrates xCTBP/xALDH1 bound T3 within living cells. These results raise the possibility that an unknown factor(s) besides NAD+ and NADH may modulate the thyroid-hormone-binding activity of xCTBP/xALDH1. In comparison, thyroid hormone, at its physiological concentration, would poorly modulate the enzyme activity of xCTBP/xALDH1. [ABSTRACT FROM AUTHOR]

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)-dependent aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes into either nonactivated acids or CoA-activated acids. The NADP-dependent nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) belongs to the first subclass. It catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate into 3-phosphoglycerate via a two step mechanism in which deacylation is rate-limiting. Recent studies on GAPN from Streptococcus mutans have shown that residue Glu268 plays an essential role only in the deacylation step [Marchal, S., Rahuel-Clermont, S. & Branlant, G. (2000) Biochemistry 39, 3327–3335]. The substitution of Glu268 by alanine or glutamine leads to mutants in which the attacking water molecule involved in the hydrolytic process is poorly activated. Activity can be restored by the presence of hydroxylamine and hydrazine. Neutral and protonated forms of both nucleophiles are recognized by the deacylating subsite of both mutants. pH rate profiles of deacylation show pKa values of 6.3 and 8.1 with hydroxylamine and hydrazine, respectively, which are those of the nucleophiles in solution. The increase in enzymatic rate is probably due to a high local concentration and not to a change of the chemical reactivity of both nucleophiles upon their binding within the active site of both mutants. The deacylation subsite of the wild-type also binds hydroxylamine and hydrazine but as inhibitors of the hydrolytic process and not as acyl acceptors. Altogether, the results point out the crucial role of the carboxyl group of Glu268 in preventing nucleophiles, other than water, from binding as efficient acyl acceptors. This may also explain why CoA-dependent ALDHs never possesses a glutamate residue at position 268. [ABSTRACT FROM AUTHOR]

Examines the occurrence of aldehyde dehydrogenase (ALDH) residue triad. Use of kanamycin for phage-infected cells; Description of ALDH purification; Effects of the hydroxyl substituent on electronegativity of the phenyl ring.

Examines the overproduction of panich betaine aldehyde dehydrogense in echeriachia coli. Synthesis of osmoprotectant glycine; Oxidation of aminoaldehyde; Stability and kinetic chractietesic characteristics.

Among pyridine-nucleotide-dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic residue, Glu140, coordinates the adenine ribose 2′ hydroxyl. In many NAD-dependent dehydrogenases a similarly placed carboxylate is thought to be responsible for exclusion of NADP. The corresponding residue in most (≈ 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD-specific enzymes. Site-directed mutagenesis was performed on this residue in rat class 3 ALDH. Our results indicate that this residue contributes to tighter binding of NAD in the native enzyme, but suggest that additional factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essential for binding both coenzymes, consistent with its conservation in nearly all ALDHs (> 98%). [ABSTRACT FROM AUTHOR]

Vitamin A (retinol) and provitamin A (β-carotene) are metabolized to specific retinoid derivatives which function in either vision or growth and development. The metabolite 11-cis-retinal functions in light absorption for vision in chordate and nonchordate animals, whereas all-trans-retinoic acid and 9-cis-retinoic acid function as ligands for nuclear retinoic acid receptors that regulate gene expression only in chordate animals. Investigation of retinoid metabolic pathways has resulted in the identification of numerous retinoid dehydrogenases that potentially contribute to metabolism of various retinoid isomers to produce active forms. These enzymes fall into three major families. Dehydrogenases catalyzing the reversible oxidation/reduction of retinol and retinal are members of either the alcohol dehydrogenase (ADH) or short-chain dehydrogenase/reductase (SDR) enzyme families, whereas dehydrogenases catalyzing the oxidation of retinal to retinoic acid are members of the aldehyde dehydrogenase (ALDH) family. Compilation of the known retinoid dehydrogenases indicates the existence of 17 nonorthologous forms: five ADHs, eight SDRs, and four ALDHs, eight of which are conserved in both mouse and human. Genetic studies indicate in vivo roles for two ADHs (ADH1 and ADH4), one SDR (RDH5), and two ALDHs (ALDH1 and RALDH2) all of which are conserved between humans and rodents. For several SDRs (RoDH1, RoDH4, CRAD1, and CRAD2) androgens rather than retinoids are the predominant substrates suggesting a function in androgen metabolism as well as retinoid metabolism. [ABSTRACT FROM AUTHOR]

Investigates the aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. Oxidation of aldehydes to carboxylic acid form; Reduction of protein with excess dithionite; Crystal structures of homologous enzymes.

Examines the mechanism of inhibition of aldehyde dehydrogenase by citral. Chemical structures of citral isomers; Inhibition of retinoic acid formation; Involvement of an E1 cytoplasmic enzyme in citral metabolism.

Examines the simulation of premature retinoic acid synthesis in Xenopus embryos of aldehyde dehydrogenase ALDH1. Production of ligand retinoic acid from vitamin A; Role of retinoic acid synthesis by analysis of retinoid signaling; Use of retinoic acid bioassay to demonstrate embryos from fertilization to gastrula stage.

Retinoic acid. a developmental signal implicated in the formation of the neural axis. is present at high levels in the early embryonic trunk region, where it is synthesized by a novel dehydrogenase. Here we show that the same enzyme is inducible by retinoic acid in P19 teratocarcinoma cells, and we report the cloning from P19 cells of a CDNA encoding a novel dehydrogenase, named retinaldehyde dehydrogenase-2 (RALDH-2). Expression in COS calls shows RALDH-2 to be highly effective in oxidation of retinaldehyde, with no detectable activity on any other aldehyde tested. In situ hybridization histochemistry on the embryonic trunk reveales RALDH-2 mRNA both in mesoderm and neuroectoderm, with highest neuroectodermal expression in the ventral horn of the spinal cord at two restricted locations along the anteroposterior axis. presumable the subpopulation of motoneurons that innervate the limbs. [ABSTRACT FROM AUTHOR]

The major proteins (crystallins) of the transparent, refractive eye lens of vertebrates are surprisingly diverse group of multifunctional proteins. A number of lens crystallins display taxon-specificity. In general, vertebrate crystallins have been recruited from stress-protective proteins (i.e. the small heat-shock proteins) and a member of metabolic enzymes by a gent-sharing mechanism. Despite the existence of refractive lenses in the complex and compound eyes of many invertebrates, relatively little is known about their crystallins. Here we review for the first time the state of knowledge of invertebrate crystallins. The major cephalopod (squid, octopus, and cuttlefish crystallins (S-crystallins) have, like vertebrate crystallins, been recruited from a stress protective metabolic enzyme, glutathione S-transferase. The presence of overlapping AP-1 and antioxidant responsive-like sequences that appear functional in transfected vertebrate cells suggests that the recruitment of glutathione S-transferase to S-crystallins involved response to oxidative stress. Cephalopods also have at least two taxon-specific crystallins: Ω-crystallin, related to aldehyde dehydrogenase, and O-crystallin, related to a superfamily of lipid-binding proteins, L-crystallin (probably identical to Ω-crystailin) is the major protein of the lens of the squid photophore, a specialized structure for emitting light. The use of L/Ω-crystallin in the ectodermal lens of the eye and the mesodermal lens of the photophore of the squid contrasts with the recruitment of different crystallins in the ectodermal lenses of the eye and photophore of fish. S- and Ω-crystallins appear to be lens-specific (some S-crystallins are also expressed in cornea) and, except for one S-crystallin polypeptide (SL11/Lops4; possibly a molecular fossil), lack enzymatic activity The S-crystallins (except SL11/Lops4) contain a variable peptide that has been inserted by exon shuffling. The only other invertebrate crystallins that have been examined are in one marine gastropod (Aplysia, a sea hare), in jellyfish and in the compound eyes of some arthropods; all are different and novel proteins. Drosocrystallin is one of three calcium binding taxon-specific crystallins found selectively in the acellular corneal lens of Drosophila, while antigen 3G6 is a highly conserved protein present in the ommatidial crystallin cone and central nervous system of numerous arthropods. Cubomedusan jellyfish have three novel crystallin families(the J-crystallins); the J1-crystallins are encoded in three very similar intronless genes with markedly different 5′ flanking sequences despite their almost identical encoded proteins and high lens expression. The numerous refractive structures that have evolved in the eyes of invertebrates contrast markedly with the limited information on their protein composition, making this field as exciting as it in underdeveloped. The similar requirement of Pax-6 (and possibly other common transcription factors) for eye development as well as the diversity, taxon-specificity and recruitment of stress-protective enzymes as crystallins suggest that borrowing multifunctional proteins for refraction by a gene sharing strategy may have occurred in invertebrates as it did in vertebrates. [ABSTRACT FROM AUTHOR]

In this report, we describe the isolation of a 4020-bp genomic Pst1 fragment of Desulfovibrio gigas harboring the aldehyde oxido-reductase gene. The aldehyde oxido-reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 50% (± 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria. [ABSTRACT FROM AUTHOR]

Human liver aldehyde dehydrogenase (E3 isozyme), with wide substrate specificity and low Km for 4-aminobutyraldehyde, was only recently characterized [Kurys, G., Ambroziak. W. & Pietruszko, R. (1989) J. Biol. Chem. 264, 4715–4721] and in this study we report on its primary structure. Polyclonal antibodies, specific for the E3 isozyme and three oligonucleotide probes derived from amino acid and sequence of the E3 protein, were used for isolation of the first cDNA clone encoding the human enzyme (1503 bp: coding for 440 amino acid residues). Additional clones were obtained by using the first isolated clone as a probe. The largest clone of 1635 bp coded for 462 amino acid residues; it was longer at the 3'end of the cDNA non-coding region. The identity of the clone was established by DNA sequencing and by comparison with peptide sequences derived from the E3 protein, which constituted approximately 29% of the total primary structure of the E3 isozyme. The start codon was never encountered despite a variety of different approaches (500 amino acid residues were expected on the basis of SDS-gel molecular-mass determination of the E3 isozyme subunit). Despite the great catalytic similarity between the E3 and E1 isozymes [Ambroziak, W. & Pietruszko, R. (1991) J. Biol. Chem. 266, 13011–13018], the primary structure of the E3 isozyme has only approximately 40.6% of positional identity with that of the E1 isozyme. Sequence comparison with GenBank and Protein Identification Resource database sequences indicated no primary structure of aldehyde dehydrogenase more closely resembling the E3 isozyme than that of Escherichia coli betaine aldehyde dehydrogenase (52.7% positional identity), a prokaryotic enzyme specific for betaine aldehyde. [ABSTRACT FROM AUTHOR]

The Gram-positive Corynebacterium glutamicum has the potential to synthesize L-lysine via a split pathway, where amino-ketopimelate is converted to the ultimate lysine precursor diaminopimelate either by reactions involving succinylated intermediates, or by one single reaction catalysed by D-diaminopimelate dehydrogenase. To quantify the flux distribution via both pathways, [sup13]C-enriched glucose was used and specific enrichments in lysine and in pyruvate-derived metabolites were determined by [sup13]C- and [sup1]H-NMR spectroscopy. Using a system of linear equations, the contribution of the D-diaminopimelate dehydrogenase pathway was determined to be about 30% for the total lysine synthesized. This was irrespective of whether lysine-accumulating mutants or the wild-type strain were analysed. However, when the distribution was determined at various cultivation times, the flux partitioning over the dehydrogenase pathway in a producing strain decreased from 72% at the beginning to 0% at the end of lysine accumulation. When ammonium sulphate was replaced by the organic nitrogen source glutamate, the ammonium-dependent D-diaminopimelate dehydrogenase pathway did not contribute to total lysine synthesis at all. Additional experiments with varying initial ammonium concentrations showed that in Corynebacterium glutamicum the flux distribution over the two pathways of lysine synthesis is governed by the ammonium availability. This is thus an example where an anabolic pathway is directly influenced by an extracellular medium component, probably via the kinetic characteristics of D-diaminopimelate dehydrogenase. [ABSTRACT FROM AUTHOR]

The Desulfovibrio gigas aldehyde oxido-reductase contains molybdenum and iron-sulfur clusters. Mössbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxydized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (ΔEQ = 0.62 ± 0.02 mm/s and δ = 0.27 ± 0.01 mm/s) typical for the [2Fe-2S]2+ state. Mössbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = ½ system. Two ferrous sites with different ΔEQ values (3.42 mm/s and 2.93 mm/ s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 ± 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters. [ABSTRACT FROM AUTHOR]

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation. Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression. These results indicate that aid is under carbon regulation mediated by at least two different mechanisms. [ABSTRACT FROM AUTHOR]

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non- activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 μM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red-Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl-Sepharose and further chromatographic separation by red-Sepharose and FPLC using Mono-Q and phenyl-Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels. [ABSTRACT FROM AUTHOR]

Allelic differences at the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase loci may have an important role in an individual's alcohol sensitivity. We have cloned and sequenced all nine exons of an ADH22 allele which codes for an ‘atypical’ ADH, ADHβ2. Our sequence data shows that the histidine at residue 47 of ADHβ2 is encoded by CAC. Surprisingly, no silent substitution was found between the coding regions of ADH21 [Duester, G., Smith, M., Bilanchone, V. & Hatfield, G. W. (1986) J. Biol. Chem. 261, 2027–2033.] and ADH22 alleles over the 1122 nucleotide sites. [ABSTRACT FROM AUTHOR]

The cDNA coding for rat liver mitochondrial aldehyde dehydrogenase was cloned and sequenced. It contained an open reading frame of 1557 bp. Of the deduced 519 amino acid residues, 19 were proposed to correspond to the signal peptide necessary to allow the protein to enter the mitochondria [Farré, J., Guan, K.-L. and Weiner, H. (1987) Biochem. Biophys. Res. Commun. 150, 1083–1087]. The sequence of the 500 amino acid residues comprising the mature subunit was 96% identical to that of the corresponding human liver mitochondrial enzyme. The longest cDNA isolated coding for the bovine liver enzyme contained sequence information corresponding to residues 72–500 of the rat or human enzyme. The deduced protein sequence of these residues was 94% identical to those of the human enzyme. Over 60% of the bases in the coding region of the rat cDNA were G and C. These residues were clustered non-randomly and two potentially stable stem-loop structures could be calculated as existing in the mRNA. One would be found in the region coding for amino acid residues 64–79; a similar secondary structure could be responsible for the existing truncated cDNAs from the bovine-cDNA libraries. Comparisons with the amino acid sequences of other aldehyde dehydrogenases support the suggestion that the NAD-binding domain may be located in the middle portion of the mature enzyme. [ABSTRACT FROM AUTHOR]

The primary structure of the mitochondriaI form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N-terminal heterogeneity in size similar to thai for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys-302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non-human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186000, consists of two subunits of equal size (Mr 92000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quinoprotein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases. [ABSTRACT FROM AUTHOR]

The 500-residue amino acid sequence of the subunit of mitochondrial human liver aldehyde dehydrogenase is reported. It is the first determined for this enzyme type from any species, and is based on peptides from treatments with trypsin, CNBr, staphylococcal Glu-specific protease, and hydroxylamine. The chain is not blocked (in contrast to that of the acetylated cytosolic enzyme form), but shows N-terminal processing heterogeneity over the first seven positions. Otherwise, no evidence for subunit microheterogeneities was obtained. The structure displays 68% positional identity with that of the corresponding cytosolic enzyme, and comparisons allow functional interpretations for several segments. A region with segments suggested to participate in coenzyme binding is the most highly conserved long segment of the entire structure (positions 194-274). Cys-302, identified in the cytosolic enzyme in relation to the disulfiram reaction, is also present in the mitochondrial enzyme. A new model of the active site appears possible and involves a hydrophobic cleft. Near-total lack of conservation of the N-terminal segments may reflect a role of the N-terminal region in signaling the transport of the mitochondrial protein chains. Non-conservation of interior regions may reflect the differences between the two enzyme forms in subunit interactions, explaining the lack of heterotetramic molecules. The presence of some internal repeat structures is also noted as well as apparently general features of differences between cytosolic and mitochondrial enzymes. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase from different origins (brain, muscle, erythrocytes) binds to microtubules polymerized from pure brain tubulin and causes bundle formation in vitro. ATP is shown to dissociate these bundles into individual microtubules, while the dehydrogenase is not displaced from the polymers by this nucleotide. ATP can be replaced by adenosine 5′-(β,γ-imido]triphosphate, a nonhydrolyzable analog of ATP. These data are interpreted in terms of dissociation of the glyceraldehyde-3-phosphate dehydrogenase tetramer into dimers by ATP. The enzyme is also efficiently purified by a tubulin-Sepharose affinity chromatography. [ABSTRACT FROM AUTHOR]

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment cause a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH: cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehydes. The enzyme Km values lie in the millimolar range for acetaldehye, propionaldehyde, benzaldehyde and phenylacetaldehye and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed. [ABSTRACT FROM AUTHOR]

The structural divergence between the cytoplasmic isoenzymes of aldehyde dehydrogenase from different species was investigated by analysis of peptides from the horse protein, and correlation of the results with the complete primary structure of the human isoenzyme. The amino acid sequences of these two proteins show a high degree of homology (91 % of residues compared are identical). The differences observed are spread over the entire polypeptide chains, with only one cluster, which is close to a reactive cysteine residue and also adjacent to the most conserved region (covering 68 residues) in the primary structures of the whole enzymes. The secondary structure predicted for the human isoenzyme is mainly unaffected by the residue differences in the horse isoenzyme, although limited conformational changes might be compatible with an unexpected over representation of differences involving isoleucine (12 of 43 exchanges represent a loss of lie in the horse protein). To cysteine residues that correlate with catalytic activity are identically positioned in the enzyme from the two species. [ABSTRACT FROM AUTHOR]

Analysis of CNBr fragments and other peptides from human liver cytoplasmic aldehyde dehydrogenase enabled determination of the complete primary structure of this protein. The monomer has an acylated amino terminus and is composed of 500 amino acid residues, including II cysteine residues. No evidence of any microheterogeneity was obtained, supporting the concept that the enzyme is a homotetramer. The disulfiram-sensitive thiol in the protein, earlier identified through its reaction with iodoacetamide, is contributed by a cysteine residue at position 302. while the cysteine which in horse liver mitochondrial aldehyde dehydrogenase is reactive with coenzyme analogs appears to correspond to either Cys-455 or Cys-463. Analysis of glycine distribution and prediction of secondary structures to localize βαβ regions typical for coenzyme-binding are not fully unambiguous, but suggest a short region around position 245 as a likely segment for this function. In this region, sequence similarities to parts of a bacterial aspartate-β-semialdehyde dehydrogenase and a mammalian alcohol dehydrogenase were noted. Otherwise, no extensive similarities were detected in comparisons with characterized mammalian enzymes of similar activity or subunit size as aldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase, respectively). [ABSTRACT FROM AUTHOR]

An NAD+-dependent alcohol dehydrogenase (ADH) of the Antarctic psychrophile Moraxella sp. TAE123 was purified to homogeneity with an overall yield of 16.7 % and further characterized. The native enzyme had an apparent molecular mass of 240 kDa and consisted of four identical 52-kDa subunits. The pI of the enzyme was determined to be 5.5, while its optimum pH is 7.5. The enzyme contained 1 zinc atom/subunit and exhibited a remarkable thermal lability. Moraxella sp. TAE123 ADH exhibited a wide range of substrate specificity similar to its mammalian counterparts and in contrast to other microbial ADHs. It oxidized mainly primary and secondary aliphatic alcohols. The highest reaction rate was observed when ethanol was used as substrate. A gradual decrease in rate was observed by increasing the length and branching of the carbon chain of the alcohol. This enzyme oxidized effectively large bulky alcohols, such as diphenylmethanol. Reduction of aldehydes and ketones was also observed. N-terminal amino acid sequence analysis of the enzyme did not reveal any similarity with the amino termini of all other ADHs, while an unexpected significant similarity was observed with the amino terminal sequence of four prokaryotic aldehyde dehydrogenases. [ABSTRACT FROM AUTHOR]

Twelve aldehyde dehydrogenase (ALDH) genes have been identified in humans. These genes, located on different chromosomes, encode a group of enzymes which oxidizes varieties of aliphatic and aromatic aldehydes. Metabolic disorders and clinical problems associated with mutations of ALDH1, ALDH2, ALDH4, ALDH10 and succinic semialdehyde (SSDH) genes have been emerged. Comparison of the human ALDHs indicates a wide range of divergency (> 80-< 15 % identity at the protein sequence level) among them. However, several protein regions, some of which are implicated in functional activities, are conserved in the family members. The phylogenic tree constructed of 56 ALDH sequences of humans, animals, fungi, protozoa and eubacteria, suggests that the present-day human ALDH genes were derived from four ancestral genes that existed prior to the divergence of Eubacteria and Eukaryotes. The neighbor-joining tree derived from 12 human ALDHs and antiquitin indicates that diversification within the ALDH1/2/5/6 gene cluster occurred during the Neoproterozoic period (about 800 million years ago). Duplication in the ALDH 3/10/7/8 gene cluster occurred in Phanerozoic period (about 300 million years ago). Separations of ALDH3/ALDH10 and that of ALDH7/ALDH8 had occurred during the period of appearance and radiation of mammalian species. [ABSTRACT FROM AUTHOR]

Fatty aldehyde dehydrogenase (ALDH) from the luminescent marine bacterium, Vibrio harveyi, differs from other ALDHs in its unique specificity and high affinity for NADP+. Two glutamic acid residues, Glu253 and Glu377, which are highly conserved in ALDHs, were investigated in the present study. Mutation of G1u253 to Ala decreased the kcat, for ALDH activity by over four orders of magnitude without a significant change in the Km values for substrates or the ability to interact with nucleotides. Both thioesterase activity and a pre-steady-state burst of NAD(P)H were also eliminated, implicating Glu253 in promoting the nucleophilicity of the cysteine residue(Cys289) involved in forming the thiohemiacetal inteiwediate in the enzyme mechanism. Mutation of Glu377 to Gin (E377Q mutant) selectively decreased the kcat, for NAD+-dependent ALDH activity (>102-fold) compared to only a 6-fold loss in NADP+- dependent activity without comparable changes to the Km, values for substrates. Consequently, the E377Q mutant had a very high specificity for NADP+(kcat/Km> 103 of that for NAD+) which was over 20 times higher than that of the wild-type ALDH. Although a pre-steady-state burst of NAD(P)H was eliminated by this mutation, thioesterase activity was completely retained. Using [1-2H]acetaldehyde as a substrate, a significant deuterium isotope effect was observed, implicating Glu377 in the hydride transfer step and not in acylation or release of the acyl group from the cysteine nucleophile. The increase in specificity of the E377Q mutant for NADP+ is consistent with a change in the rate-limiting step determining kcat, from nucleotide-dependent NAD(P)H dissociation to hydride transfer. The results provide biochemical evidence that the two highly conserved Glu residues are involved in different functions in the active site of V. harveyi ALDH. [ABSTRACT FROM AUTHOR]

A fragment of a cytosolic thyroid-hormone-binding protein from Xenopus liver was reported to be 92–100% identical to residues 236–258 in several cytosolic aldehyde dehydrogenases [Yamauchi, K. & Tata, J. R. (1994) Eur. J. Biochem. 225, 1105–1112], which have been proposed to form part of the hinge region necessary to bind the adenosine moiety of NAD. Here we investigated the effects of two thyroxine analogs, 3,3′,5-triiodo-L-thyronine and 3,3′,5-triiodothyroacetic acid, on purified human liver mitochondrial and cytosolic aldehyde dehydrogenases. The compounds were found to be competitive inhibitors against NAD and uncompetitive inhibitors with respect to aldehyde. At pH 7.4, the apparent Ki values were in the micromolar range when the concentration of NAD was varied. The inhibition against recombinantly expressed mutant forms of aldehyde dehydrogenase, which possessed diminished NAD binding, was determined. Essentially no differences were found between the native enzyme and the mutants, showing that the analog binding was not affected by altering the NAD-binding site. Furthermore, the analogs could displace NAD but not NADH from the enzyme. These findings indicated that the binding of NAD differed from that of NADH, and that aldehyde dehydrogenases, like other dehydrogenases, can be inhibited by thyroxine analogs. [ABSTRACT FROM AUTHOR]

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CY P), including CY P1A1/2, CY P2B1/2, CY P2C6, CY P2E1, CY P3A1/2, glutathione transferase α (GSTα), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CY P1A1/2, CY P2B1/2, CY P3A1/2, GSTa, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CY P2C6 and CY P2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CY P1A1/2 (13.6-fold) and GSTa (3.9-fold) mRNA expression in 3-methyl cholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CY P2C6 (10-fold), CY P3A1/2 (11.2-fold), GSTα (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CY P2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CY P transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2–3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CY P2B1 or CY P2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes. [ABSTRACT FROM AUTHOR]

A series of NAD+ analogues, modified on the pyridinium ring, have been tested for their enzymic properties in reactions with D-glyceraldehyde-3-phosphate dehydrogenase form sturgeon muscle, rabbit muscle and c stearothermophilus. The observed activity, inhibition and binding data are correlated to the structure of the enzyme and coenzyme analogue by model building on a Vector General inter live graphic display system using coordinates from the B. stearothermophilus holoenzyme structure. Most of the analogues with substituents in the pyridinium-3 position could be bound to glyceraldehyde-3- phosphate dehydrogenase, either in manner similar to NAD+ or in a completely different way with the substituted ridinium ring rotated l10° or more around the glycosidic bond. This indicates different possible modes of binding of NAD+ analogues within the pyridinium binding subsite. Analogues with substituents in the pyridinium-4 position are shown to be weakly bound to glyceraldehyde-3- phosphate dehydrogenase. This is explained by a strong interaction of the substituent in the 4 position with the residues Asn-313 and Cys-149. [ABSTRACT FROM AUTHOR]

1. Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the Mitochondrial enzyme by isoelectric focusing. 2. Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pI = 5.22 ± 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pI = 5.22 and five further bands of pI 5.48 ± 0.09, 5.56 ± 0.07, 5.65 ± 0.06, 5.70 ± 0.03 and 5.76 ± 0.02. Possible origins of the isoenzymes are discussed. 3. Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram-modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group. 4. The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4-nitrophenoxide ion during the hydrolysis of 4-nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 ± 1 s-1 and the steady-state rate constant was 0.2 s-1, values similar to those previously reported for the cytoplasmic enzyme. 5. The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s-1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady-state mechanism is discussed. [ABSTRACT FROM AUTHOR]

Reports on kinetic studies with rat-brain succinic-semialdehyde dehydrogenase. Enzyme purification; Exhibition of a relatively low Km value for succinic semialdehyde; Inhibition by p-hydroxybenzaldehyde; Metabolism of aldehydes.

The kinetic mechanism for NADP-dependent isocitrate dehydrogenase from the digestive gland of the mussel Mytilus edulis has been investigated. Initial-rate studies and substrate-analogue and product-inhibition patterns are consistent with a rapid-equilibrium random-ordered reaction mechanism, both with respect to substrate addition and product release. Product inhibition by NADPH is non-competitive versus D-isocitrate at sub-saturating concentrations of NADP, and competitive with respect to NADP at all D-isocitrate concentrations. 2-Oxoglutarate inhibition is competitive versus D-isocitrate at all NADP concentrations. The inhibition by 2-oxoglutarate versus NADP is either non-competitive or uncompetitive. Tricarballylic acid (a substrate analogue of D-isocitric acid) gives competitive inhibition versus D-isocitrate at all NADP levels and mixed inhibition versus NADP, at sub-saturating levels of D-isocitrate. [ABSTRACT FROM AUTHOR]

A threefold decrease in specific activity of glycerol-3-phosphate dehydrogenase was found on going from 800 nM to 10 nM enzyme concentration. According to ultracentrifugal analyses the dimeric glycerol-3-phosphate dehydrogenase (molecular weight 78000) dissociates into monomers in the equilibrium mixture of its substrates and products. The concentration-dependent decrease in the specific activity is interpreted as a consequence of subunit dissociation and the estimated dissociation constants are 0.7 μM and 3.5 μM at 38 °C and 20 °C respectively. According to active-enzyme-band centrifugation experiments and kinetic analysis aldolase forms a complex with glycerol-3-phosphate dehydrogenase and this complex formation influences the specific activity of the dehydrogenase. The interaction between glycerol-3-phosphate dehydrogenase and aldolase can provide a regulatory mechanism at the branching point of glycol and lipid metabolic pathways. [ABSTRACT FROM AUTHOR]

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with specific activity of 380 nmol NADH min-1 mg-1. An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (λexcit = 290 nm, λemission = 340 nm) is quenched on the binding of NAD+ and NADPH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilized to determine the dissociation constant for the enzyme · NADH complex (Kd = 1.2 ± 0.2 μM). A Hill plot of the data gave a straight line with a slope of 1.0 ± 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weight were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212000 ± 8000 and 205000 respectively. Each enzyme consisted of four subunits with molecular weight of 53000 ± 2000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases. [ABSTRACT FROM AUTHOR]

Levels of liver alcohol dehydrogenase, liver mitochondrial aldehyde dehydrogenase and brain (Na + K)-stimulated ATPase were determined in control rats and in rats fed an ethanol liquid diet for 7 weeks, The alcohol dehydrogenase level did not change with the diet; however, the levels of mitochondrial aldehyde dehydrogenase and of the (Na + K)-ATPase were increased 2.5-fold and 2-fold respectively. Also, the levels of these enzymes from brain and liver fractions of rats given a 20% ethanol solution as the sole drinking fluid were determined at intervals from 4 to 18 weeks. A progressive increase was found in the level of aldehyde dehydrogenase and of ATPase. To ascertain whether synthesis or degradation of these enzymes were responsible for the marked increase in activity the turnover of several liver fractions and of the indicated enzymes were measured by the dual-labelled isotope technique. Long-term ethanol administration did not affect the rate of turnover of the liver cell fractions except for the mitochondrial fraction. This fraction showed a marked decrease in the rate of degradation. The degradation constant of the liver alcohol dehydrogenase did not change; however, the rate of degradation of liver mitochondrial aldehyde dehydrogenase (low-Km) and of brain (Na + K)-ATPase was decreased. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220000 ± 10000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 µM, 170 gM and 130 µM, respectively. [ABSTRACT FROM AUTHOR]

Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin-assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20 % (spec. act. with butanal 0.33 kat/kg) was NAD-dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 kat/kg) was NADP-dependent, It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are 'constitutive' and their physiological function is uncertain. When the bacteria were grown on n-paraffin a new membrane-bound NAD-dependent aldehyde dehydrogenase activity was produced. [ABSTRACT FROM AUTHOR]

Sturgeon glyceraldehyde-3-phosphate dehydrogenase has been strongly adsorbed on an 8-azolinked NAD+ -Sepharose derivative. Specific elution of the matrix-bound enzyme was obtained with relatively low concentrations of NAD+ (20-100 µM) in elution buffer. Recovery of adsorbed enzyme amounted to 90-95% when an hydrophilic 1,3-diaminopropan-2-ol spacer arm was used instead of the more conventional diaminohexyl derivative. A capacity of 0.8 mg of sturgeon enzyme per g of derivative was estimated either under batchwise conditions or by frontal chromatography. Under similar conditions, purified apoglyceraldehyde-3-phosphate dehydrogenase from yeast did not bind significantly to the same NAD+ derivative. Crude extracts of sturgeon muscle can be purified more than 15-fold upon chromatography on this derivative. Unlike the sturgeon enzyme, yeast glyceraldehyde-3-phosphate dehydrogenase was significantly adsorbed to an N6(6-aminohexyl)-AMP derivative under chromatographic or batchwise conditions. At least 40 units (i.e. 0.2-0.3 mg) of yeast glyceraldehyde-3-phosphate dehydrogenase can be bound per g of matrix. An 8-fold purification of the enzyme can be obtained upon specific elution with NAD+. Relatively large amounts of yeast enzyme (i.e. up to 100 mg) can be partially purified by this one-step procedure. The distinct adsorption properties of yeast and sturgeon glyceraidehyde-3-phosphate dehydrogenases are discussed in the light of the known cooperative coenzyme binding properties of these enzymes. The relevance of affinity chromatography to the preparation of sizeable amounts of glyceraldehyde-3-phosphate dehydrogenase is discussed and compared with conventional procedures. Possible interference of glyceraldehyde-3-phosphate dehydrogenase with the 'general ligand' binding of other dehydrogenase and kinases in crude extracts is discussed. [ABSTRACT FROM AUTHOR]

1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or kcat. Some buffer anion effects were noted. 2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2-bromo-2-phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate. 3. Values for the rate of binding and dissociation of NAD+ were obtained from the steady-state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases. 4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25s-1 being obtained for Kcat No isotope effect was observed on V using [1-²H]propionaldehyde as substrate suggesting that NADH release might be rate-limiting in the steady-state. 5. The implications of the non-linear steady-state behaviour are discussed. [ABSTRACT FROM AUTHOR]

1. The dimeric flavoprotein pig heart lipoamide dehydrogenase (NADH: lipoamide oxido-reductase, EC 1.6.4.3) has been polythiolated by reaction with N-acetylhomocysteine thiolactone at pH 10.5 for 24 h. Under these conditions 5–6 mol additional thiol groups are introduced per tool FAD. 2. The free polythiolated enzyme has a 50-60% lower specific activity for lipoamide, a reduced affinity for a specific anti-lipoamide dehydrogenase antibody but an unchanged apparent Km (lipoamide). The free polythiolated enzymes containing 6 mol -SH/mol FAD and 7 mol -SH/mol FAD were 270% and 640% respectively more stable to thermal inactivation at 90°C than the free native enzyme. 3. Immobilisation of the polythiolated enzyme to thiolated 6-aminohexyl-Sepharose reduced the specific activity with lipoamide to less than 10% of that of the free native enzyme, raised the apparent Km (lipoamide) to 8.3 mM and lowered its affinity for the specific antibody. The thermal stability at 90°C was enhanced by up to 25-fold. 4. Immobilisation of the polythiolated enzyme to a short spacer group, L-cysteinyl-Sepharose, reduced the specific activity with lipoamide but enhanced the stability at 90°C and in 70% (v/v) dioxane by 800% and 770% respectively relative to the free native enzyme. These data are discussed in terms of the effects of proximity to the matrix backbone. 5. The marked improvement in stability of the polythiolated enzyme was matched by lipoamide dehydrogenase immobilised directly to CNBr-activated Sepharose at pH 7.5 and pH 9.0. However, in this case the specific activity of the immobilised enzyme was 300-350% less than that of the poly-thiolated enzyme. These data are discussed in terms of multiple attachment of the enzyme to the matrix and the possibility of disulphide cross-links in the polythiolated enzyme. [ABSTRACT FROM AUTHOR]

1. Pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose by thiol-disulphide interchange via a series of thiolated spacer molecules of increasing length. A number of properties of the immobilised enzyme have been investigated in order to ascertain the effects of proximity to the matrix backbone. 2. Proximity to the matrix backbone reduced the specific activity for lipoamide as substrate but enhanced by 3–8-fold the diaphorase activity with 2,6-dichloroindophenol. These observations are explained in part by an increase in the apparent Km for lipoamide when the enzyme is covalently attached to Sepharose via a short spacer molecule. 3. Both the thermal stability at 90°C and the stability in 30% (v/v) dioxane are enhanced by up to 200% when the enzyme resides close to the matrix but approach those of the native enzyme as the length of the spacer molecule is increased. 4. These data have been correlated with measures of the accessibility of the enzyme as the nominal length of the spacer arm was increased. Thus, as the chain length increased, the rate of cleavage of the disulphide linkage between the enzyme and spacer increased and the enzyme became more susceptible to proteolysis by thermolysin. In contrast, increasing the chain length of the spacer made the enzyme less amenable to inhibition by a specific antibody. 5. These data are discussed in terms of the effect of the matrix on the conformation of the bound enzyme. [ABSTRACT FROM AUTHOR]

The inhibition of rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase by specific antibodies produced in rabbits has been studied. The results suggest that no influence on the enzyme active site is caused by the interaction with antibody, the inhibition being due entirely to the restricted accessibility for substrates of a part of dehydrogenase molecules included in the immune precipitate. Soluble complexes of the enzyme with monovalent Fab antibody fragments retain full catalytic activity. Modification of 8 -SH groups per mole of glyceraldehyde-3-phosphate dehydrogenase with p-chloromercuribenzoate results in no alterations in the quantitative precipitin curve, thus supporting the conclusion about the different localization of species-specific antigenic determinants of the enzyme and its active center. Interaction with monovalent Fab fragments of antibody stabilizes the structure of the dehydrogenase. Eight molar equivalents of Fab fragments almost completely protect the enzyme from cold inactivation in the presence of 0.15 M NaCl. Complex formation with Fab fragments does not prevent, however, the ADP-induced inactivation of the enzyme. [ABSTRACT FROM AUTHOR]

The reactivity and the mode of activation of the essential - SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rote of Cys-149 conform to doubly sigrnoid curves. All these curves implicate two ionizing groups (pK1 = 5.5, pK2 = 8.2). It is concluded that there are two reactive forms of the -SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mereaptide ion. The two reactive forms are transformed into each other with pK2 = 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 = 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists m a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD. [ABSTRACT FROM AUTHOR]

1. The enzyme kinetics of semi-purified pig brain aldehyde dehydrogenase have been investigated. 2. The form of the basic double reciprocal plots and plots in the presence of known concentrations of NADH are consistent with a compulsory order reaction pathway in which NAD+ is bound first, followed by the aldehyde. The products are released in a fixed order, acid first then NADH. 3. The overall reaction is irreversible, and proceeds to completion. Acid products are not inhibitory. This is consistent with the release of acid products being irreversible. 4. The form of inhibition by high aldehyde concentrations is consistent with the proposed reaction pathway. 5. Apparent substrate activation is observed with acetaldehyde and propionaldehyde. [ABSTRACT FROM AUTHOR]

The NAD-linked aldehyde dehydrogenase from pig brain has been purified some 90-fold over extracts of acetone powders of whole brain. This preparation of enzyme is unstable, and a less pure, more stable preparation has been used to investigate some of the properties of the enzyme. Inhibition of the enzyme by the monoamine oxidase inhibitors 2-phenylethylhydrazine and 2-phenyl-1-methylethylhydrazine is reported. Ethanol and reserpine have both been found not to inhibit aldehyde dehydrogenase. On the basis of inhibition by o-iodosobenzoate and phenylarsenious oxide it is proposed that the enzyme has two juxtaposed sulphydryl groups at or near the active site. Irreversible inhibition by certain conjugated, unsaturated aldehydes is reported. Non-enzymic reactions of NAD+ with indole-3-acetaldehyde and phenylacetaldehyde are described. The reaction products are spectrally similar to NADH, but by enzymic criteria are not NADH. It is postulated that the products are the adducts between the aldehydes and the nicotinamide moiety of the coenzyme. [ABSTRACT FROM AUTHOR]