Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice

Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.     

The microsomal ethanol oxidizing system mediates metabolic tolerance to ethanol in deermice lacking alcohol dehydrogenase

Metabolic tolerance to ethanol has been attributed to enhanced mitochondrial reoxidation of reducing equivalents produced in the alcohol dehydrogenase (ADH) pathway or to non-ADH mechanisms. To resolve this issue, deermice lacking low Km hepatic ADH were fed for 2 weeks a liquid diet containing ethanol or isocaloric carbohydrate and hepatocytes were isolated. Ethanol (50 mM) oxidation increased (9.8 vs 4.5 nmol/min/10(6) cells in controls). To differentiate which of two non-ADH pathways (the microsomal ethanol oxidizing system (MEOS) or catalase) was responsible for the induction, four approaches were used. First, MEOS was assayed in hepatic microsomes and found to be increased (24.4 vs 6.8 nmol/min/mg protein in controls). Second, hepatocyte ethanol metabolism was measured after addition of the catalase inhibitor azide (0.1 mM) and found to be unchanged. By contrast, the competitive MEOS inhibitor, 1-butanol, depressed metabolism in a concentration-dependent manner. A third approach relied on measurement of isotope effects known to be different for MEOS and catalase. From the isotope effect values, MEOS was calculated to contribute 85% or more of total ethanol oxidation by cells from both ethanol-fed and control animals. A fourth approach involved in vivo pretreatment with pyrazole (300 mg/kg/day for 2 days), which reduced peroxidation by catalase to 13% of control values in liver homogenates while inducing MEOS activity to 152% of controls. Hepatocytes from pyrazole-treated deermice showed a 47% increase in ethanol metabolism, paralleling the MEOS induction and contrasting with the catalase suppression. These results indicate that since metabolic tolerance occurs in the absence of ADH, it is not necessarily ADH mediated, and further, that MEOS rather than catalase accounts for basal ethanol metabolism and its increase after chronic ethanol treatment.     

Characterization of alcohol dehydrogenase in young soybean seedlings

Molecular properties of alcohol dehydrogenase (ADH) were examined in young soybean seedlings. Soybean radicle tissue is ADH-rich. Enzyme specific activity decreases slowly with the development of roots and becomes almost undetectable when the first true leaves appear. Soybean ADH was not found to be inducible by flooding. 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment increased ADH specific activity as much as 14-fold. Only one ADH isozyme was detected by isoelectric focusing. By DNA-DNA hydridization, soybean ADH genomic sequences were shown to be partly homologous to maize ADH1 cDNA. The presence of more than one Adh gene in soybean is discussed.     

Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings

Abstract Aerobically germinated seedlings of rice and Echinochloa were found to survive when placed in an anaerobic environment for 4 d, whereas pea and maize seedlings did not. Although root and shoot growth were inhibited in rice and Echinochloa under anaerobiosis, growth resumed when the seedlings were returned to aerobic conditions. Alcohol dehydrogenase (ADH) activity increased more, and protein synthesis was greater, in the shoots than in the roots under anaerobic conditions. These results suggest that, in anaerobiosis-tolerant species, ADH activity and protein synthesis in the shoots represents or results from metabolic adaptations to low oxygen. These results are discussed in terms of plant establishment and growth in a low-oxygen environment.     

Effect of anaerobiosis on alcohol dehydrogenase in oat seedlings

In order to clarify the induction of alcohol dehydrogenase (ADH) by anaerobiosis in oat (Avena sativa L.), the seedlings were exposed to anaerobiosis and activity of ADH and ADH isozyme profiles were determined. The anaerobiosis increased ADH activities in shoots and roots of the seedlings. By day 2, the activity increased 5 and 4 times in the roots and the shoots, respectively, compared with those under aerobic condition. Based on nondenaturing electrophoresis, ADH isozyme composition analysis revealed six bands consisting of a dimmer enzyme with submits encoded by three different Adh genes. Changes in staining intensity of the isozymes indicated that the increase in ADH activity in oat under anaerobiosis resulted from increased enzyme synthesis.     

Inactivation of alcohol dehydrogenase by 3-butyn-1-ol

Horse liver and yeast alcohol dehydrogenases are rapidly inactivated during their catalysis of the oxidation of 3-butyn-1-ol. In the case of the horse liver enzyme, the inactivation is secondary to covalent modification of the apoenzyme by an electrophilic product that accumulates in the reaction solution and that can also react with water, glutathione, and other enzymes. The modified protein exhibits enhanced ultraviolet absorbance, which is not bleached upon dialysis of the denatured enzyme at pH 7.4 for 24 h. The inactivation by 3-butyn-1-ol is more rapid than that which is afforded by the related alcohols 2-propyn-1-ol and 2-propen-1-ol under identical conditions and no inactivation is seen upon incubation with 3-hydroxypropanoic nitrile plus nicotinamide-adenine dinucleotide.     

Anoxia tolerance and alcohol dehydrogenase activity in lettuce seedlings

In order to clarify the effect of protein synthesis inhibition on anoxiatolerance, lettuce (Lactuca sativa L.) seedlings weresubjected to anoxic stress in the presence of cycloheximide (CHM). Atconcentrations greater than 3 , CHM decreased thesurvivability of the roots and the survivability decreased with increasing CHMdoses. At 100 CHM, the survivability was 41% of thatof non-CHM-treated seedlings. Alcohol dehydrogenase (EC 1.1.1.1) activity andATP concentration in the roots of the seedlings were also decreased by CHM,which may be one of the causes of the reduced anoxia tolerance of the seedlings.     

Identification of pristanal dehydrogenase activity in peroxisomes: conclusive evidence that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which, due to the methyl-group at the 3-position, can not undergo beta-oxidation unless the terminal carboxyl-group is removed by alpha-oxidation. The structure of the phytanic acid alpha-oxidation machinery in terms of the reactions involved, has been resolved in recent years and includes a series of four reactions: (1) activation of phytanic acid to phytanoyl-CoA, (2) hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, (3) cleavage of 2-hydroxyphytanoyl-CoA to pristanal and formyl-CoA, and (4) oxidation of pristanal to pristanic acid. The subcellular localization of the enzymes involved has remained enigmatic, with the exception of phytanoyl-CoA hydroxylase and 2-hydroxyphytanoyl-CoA lyase which are both localized in peroxisomes. The oxidation of pristanal to pristanic acid has been claimed to be catalysed by the microsomal aldehyde dehydrogenase FALDH encoded by the ALDH10-gene. Making use of mutant fibroblasts deficient in FALDH activity, we show that phytanic acid alpha-oxidation is completely normal in these cells. Furthermore, we show that pristanal dehydrogenase activity is not fully deficient in FALDH-deficient cells, implying the existence of one or more additional aldehyde dehydrogenases reacting with pristanal. Using subcellular localization studies, we now show that peroxisomes contain pristanal dehydrogenase activity which leads us to conclude that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes.     

Induction of alcohol dehydrogenase by plant hormones in alfalfa seedlings

Six-day-old alfalfa (Medicago sativa L.) seedlings were treated with auxin, abscisic acid (ABA), cytokinin and gibberellin to determine the effect of these plant hormones on induction of alcohol dehydrogenase (ADH). The ADH activity was increased at concentrations greater than 1 M for auxin and ABA and 3 M for cytokinin, respectively, and all increases were found within 6 h after treatments. However, ADH activity remained almost unchanged in the seedlings treated with gibberellin. At 100 M doses, the activities in the seedlings were 4.0-, 3.6- and 2.1-fold greater than that of non-treated seedlings for auxin, ABA and cytokinin, respectively.     

Expression of alcohol dehydrogenase in rice embryos under anoxia

Summary Alcohol dehydrogenase (ADH) activity was present in roots and shoots of 48-h rice embryos and rose in response to anoxia. The increase was accompanied by changes in the ADH isozyme pattern. Translatable levels of mRNA for two ADH peptides increases as early as 1 h after the beginning of anoxic treatment. Adh mRNA was detected in aerobically grown rice embryos by hybridization to maize Adh1 cDNA: its level increased significantly after 3 h of anoxia.     

Liver alcohol dehydrogenase inhibition by fatty acid amides, N-alkylformamides and monoalkylureas.

With ethanol as substrate, N-n-alkylformamides and mono-n-alkylureas, like fatty acid amides, inhibited horse liver alcohol dehydrogenase uncompetitively, presumably by forming ternary complexes with the enzyme - reduced nicotinamide adeninedinucleotide binary complex. Compounds with 11- or 12-atom chains were better inhibitors than longer or shorter chain compounds. $\text{In vivo}$ (mice), the urea derivatives were ineffective, as were the amides, with the exception of butanamide; the latter compound was less active than iso-butanamide. Formamides with 4- to 12-atom chains were active $\text{in vivo}$ but, unlike $\text{in vitro}$, the shorter chain compounds were the most potent. A variety of branched-chain alkyl-, cyclic alkyl- and arylalkylamides and N-substituted formamides also inhibited alcohol dehyrogenase $\text{in vitro}$.     

Wounding stress induces alcohol dehydrogenase in maize and lettuce seedlings

To understand the effect of wounding stress on alcohol dehydrogenase(ADH, EC 1.1.1.1) in monocotyledonous and dicotyledonous plants, maize(Zea mays L.) and lettuce (Lactucasativa L.) seedlings were subjected to wounding stress and ADHactivity and abscisic acid (ABA) concentration were determined. In response tothe stress, the ADH activity in seedlings of both species increased rapidly asaresult of increased synthesis of the ADH. At 12 h after thestress,the activities in the wounded lettuce and maize seedlings, respectively,increased to 1.7- and 1.5-fold of that in non-stressed seedlings. Woundingstress also increased the concentration of endogenous ABA during the first 6h in both seedlings. The maximum increased levels of ABA in thelettuce and maize seedlings were 4.9- and 4.7-fold of that in the non-stressedseedlings, respectively.     

Membrane fatty acid composition of tissues is related to body mass of mammals

Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (2263) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of 3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 2263. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.This work was supported by an Australian Commonwealth Postgraduate Research Scholarship from the University of Wollongong to P. Couture and by a grant from the Australian Research Council to A.J. Hulbert. We wish to thank Voytek Mantaj for technical assistance.     

Oleic acid is the main fatty acid related with carotenogenesis in Dunaliella salina

The variation of the fatty acid profile and the carotene content of Dunaliella salina in response to irradiance (80, 128, 640, 1000, 1500 μmol photon m⁻² s⁻¹) and nitrogen starvation were analysed. The highest fatty acid content per cell and the least polyunsaturated fatty acid percentage were exhibited under 1500 μmol photon m⁻² s⁻¹. Furthermore, the oleic acid (18:1) content maintained a positive and significant correlation with the carotene content per cell and with the irradiance. The composition of the carotene globules in Dunaliella salina may be the main determinant of this correlation. This revised version was published online in June 2006 with corrections to the Cover Date.