Characterization Of Aldehyde Dehydrogenase Research Articles

Identification and characterization of aldehyde dehydrogenase (ALDH) gene superfamily in garlic and expression profiling in response to drought, salinity, and ABA

In response to biotic and abiotic stressors, aldehydes are detoxified and converted to carboxylic acids by aldehyde dehydrogenases (ALDHs), which are enzymes that use NAD+/NADP+ as cofactors. Garlic (Allium sativum L.) has not yet undergone a systematic examination of the ALDH superfamily, despite the genome sequence having been made public. In this investigation, we identified, characterized, and profiled the expression of the garlic ALDH gene family over the entire genome. The ALDH Gene Nomenclature Committee (AGNC) classification was used to classify and name the 34 ALDH genes that were discovered. Except for chromosome 8, all AsALDH genes were dispersed across the chromosomes. AsALDH genes have various localizations, according to predictions about subcellular localization. The AsALDH proteins are more varied and closely related to rice than to Arabidopsis, according to a study of conserved motifs and phylogenetic relationships. The presence of stress modulation pathways is indicated by the abundance of stress-related cis-elements in the AsALDH genes' promoter regions. Analysis of the RNA-seq data showed that AsALDHs expressed differently in various tissues and at various developmental stages. Nine AsALDHs were chosen for study using RT-qPCR, and the results revealed that the majority of the genes were upregulated in response to ABA and downregulated in response to salinity and drought. The results of this study improved our knowledge of the traits, evolutionary background, and biological functions of AsALDHs genes in growth and development.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy

ObjectiveThe cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDHhigh) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDHhigh cells in ovarian cancer. MethodsWe compared ALDHhigh to ALDHlow cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ResultsALDHhigh cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDHhigh cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDHhigh cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. ConclusionsBased on functional characterization, ALDHhigh ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes.

Identification and characterization of aldehyde dehydrogenase 9 from Lampetra japonica and its protective role against cytotoxicity.

Aldehyde dehydrogenases (ALDHs), which oxidize aldehyde to corresponding acids, play a major role in the detoxification of various endogenous and exogenous aldehydes. In this study, we cloned and characterized ALDH9 (designated LjALDH9) from Arctic lamprey Lampetra japonica. The open reading frame of LjALDH9 was 1566 bp, encoding 521 amino acids with a predicted molecular mass of 55.68 kDa. LjALDH9 protein had a signal peptide and Aldedh domain with the active site Cys315. In addition, LjALDH9 shares high sequence homology with ALDH9 of jawed vertebrates. Real-time quantitative PCR revealed that LjALDH9 was highly expressed in the buccal gland. A reactive LjALDH9 protein was obtained by prokaryotic expression, two-step-denaturing and refolding and affinity purification. During enzyme activity analysis of recombinant LjALDH9, we found that the most suitable reaction conditions were pH7.0, 16-23 °C and Mn(2+) as the activator. Our study provides theoretical proof that LjALDH9 plays an important role in the parasitic life phase of lamprey.

Purification and characterization of aldehyde dehydrogenase with a broad substrate specificity originated from 2-phenylethanol-assimilating Brevibacterium sp. KU1309

Phenylacetaldehyde dehydrogenase (PADH) was purified and characterized from Brevibacterium sp. KU1309, which can grow on the medium containing 2-phenylethanol as the sole carbon source. This enzyme was a homotetrameric protein with a subunit of 61 kDa. The enzyme catalyzed the oxidation of aryl (benzaldehyde, phenylacetaldehyde, 3-phenylpropionaldehyde) and aliphatic (hexanal, octanal, decanal) aldehydes to the corresponding carboxylic acids using NAD(+) as the electron acceptor. The PADH activity was enhanced by several divalent cationic ions such as Mg(2+), Ca(2+), and Mn(2+). On the other hand, it was inhibited by SH reagents (Hg(2+), p-chloromercuribenzoate, iodoacetamide, and N-ethylmaleinimide). The substrate specificity of the enzyme is compared with those of various aldehyde dehydrogenases.

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1

The hd-ald gene encoding aldehyde dehydrogenase ( hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD +-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K m values of the enzyme were 6.4 μM for acetaldehyde, 4.2 μM for hexanal, 2.8 μM for octanal, and 0.84 μM for decanal, whereas the k cat values for these substrates were nearly equal (51–64 min −1). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondrial matrix

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified to homogeneity from Sprague-Dawley rat liver mitochondrial matrix; its specific activity with propionaldehyde (1 mM at pH 9.0) is 1.4 mumol/min/mg. It has a native molecular weight of ca. 260,000 daltons, a subunit weight of 54,000 daltons and separates into two bands on isoelectric focusing (pI, 5.15 and 5.30); its extinction coefficient at 280 nm for 1 mg/ml solution is 1.2 and 280/260 nm ratio is 1.6. The enzyme prefers NAD over NADP as the coenzyme; the Km for NADP (67,000 microM) is three orders of magnitude greater than that for NAD (61 microM); the Km for acetaldehyde is 2 microM and for propionaldehyde is 0.8 microM at pH 7.0. The enzyme is reversibly inhibited by chloral (Ki = 3 microM) but is resistant to disulfiram inhibition. In addition another aldehyde dehydrogenase, first observed in the mitochondrial matrix during isoelectric focusing, has been partially purified. It has a Km for propionaldehyde of 0.7 mM and an isoelectric point of 6.4. Its activity with glutamic-gamma-semialdehyde (Km = 0.13 mM) is ca. 20 times higher than with propionaldehyde identifying the enzyme as glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12).

Initial Characterization of Aldehyde Dehydrogenase from Rat Testis Cytosol

Aldehyde dehydrogenase was purified 187-fold from cytosol of rat testis by chromatographic methods and gel filtration with a yield of about 50%. The enzyme exhibits absolute requirement for exogenous sulfhydryl compounds and strong dependence on temperature. Addition of 0.4mM Ca2 or Mg2 ions results in 50% inhibition. Optimally active at pH 8.5 and 50 degrees C, aldehyde dehydrogenase displays broad substrate specificity; saturation curves with acetaldehyde and propionaldehyde are non-hyperbolic, with Hill coefficients comprised between 0.8 and 0.7. Strong substrate inhibition can be observed with both aromatic and long-chain alyphatic aldehydes. According to mathematical models, Km decreases from 246 microM for acetaldehyde to 4 microM for capronaldehyde and Ki decreases from about 4mM for butyraldehyde to 0.2 mM for capronaldehyde.

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria

Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent aldehyde dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity Chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P) + analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg 2+ and K + ions were also investigated.

Purification and characterization of aldehyde dehydrogenase from human brain

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme ( N. J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta 483, 35–45 ). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 μmol/mg/min for the Peak I and 3.0 μmol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 μmol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring duringhepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 110 kDa dimer composed of 54-kDa subunits, prefers NADP + as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusions, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.

Partial purification and characterization of aldehyde dehydrogenase from sweet potato roots.

Two kinds of aldehyde dehydrogenases were found in the supernatant fraction of extracts of sweet potato roots. These enzymes had different >Km values for acetaldehyde. The major enzyme, which had a low >Km value, was partially purified from fresh sweet potato roots by several steps, including phenyl-Sepharose column chromatography. Purification increased the specific activity 240-fold compared to that at the ammonium sulfate fractionation step. The molecular weight was calculated to be close to 150,000. In potassium phosphate buffer the optimal pH was approximately 8.0, and, in Tris-HCl buffer, it was approximately 8.8. The >Km value for acetaldehyde was 7.0 µM. According to the data obtained from phenyl-Sepharose column chromatography, the enzyme was rather amphipathic in spite of its being a soluble enzyme.

Purification and characterization of aldehyde dehydrogenase from bovine liver.

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 220 000 +/- 10 000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 micron, 170 micron and 130 micron, respectively.