Acid Dehydratase Research Articles

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

The lead (Pb) lining of agriculture‐related subsidies: enhanced Golden Eagle growth rates tempered by Pb exposure

AbstractSupplementary food resources (e.g., subsidies) associated with agriculture can benefit wildlife species, increasing predictability and availability of food. Avian scavengers including raptors often utilize subsidies associated with both recreational hunting and pest shooting on agricultural lands. However, these subsidies can contain lead (Pb) fragments if they are culled with Pb‐based ammunition, potentially leading to Pb poisoning and physiological impairment in wildlife. Nesting Golden Eagles (Aquila chrysaetos) commonly forage in agricultural lands during the breeding season, and therefore, both adults and their nestlings are susceptible to Pb exposure from scavenging shot wildlife. We assessed drivers of Pb exposure in 258 nestling Golden Eagles (401 total blood samples), along with physiological and growth responses, in agricultural lands across four western states in the United States. We also evaluated the birds’ Pb stable isotope signatures to inform exposure sources. Twenty‐six percent of Golden Eagle nestlings contained Pb concentrations associated with subclinical poisoning for sensitive species (0.03–0.2 μg/g ww), 4% had Pb concentrations that exceeded subclinical poisoning benchmarks (0.2–0.5 μg/g ww), and <1% exceeded either concentrations associated with clinical poisoning (0.5–1.0 μg/g ww) and or those deemed to cause severe clinical poisoning (>1.0 μg/g ww). Lead concentrations were highest in nestlings with close proximity to fields that potentially provided subsidies and declined exponentially as distance to subsidies increased. However, close proximity to agriculture, and presumably subsidies, positively influenced nestling growth rates. Across the range of Pb exposure, nestlings experienced a 67% reduction in delta‐aminolevulinic acid dehydratase (δ‐ALAD) activity, suggesting nestlings may have been anemic or experiencing cellular damage. Isotopic ratios of 206Pb/207Pb increased non‐linearly with increasing blood Pb in Golden Eagle nestlings, and 45% of the birds were consistent with those of ammunition. However, above 0.10 μg/g ww, the proportion associated with ammunition increased to 89% of the nestlings. An improved understanding of how these positive (growth) and negative (physiology) effects associated with proximity to subsidies interact would be beneficial to managers when considering management scenarios and potentially evaluating any measures taken to reduce Pb exposure across the landscape.

Social Determinants of Health: Addressing Barriers Through Screening, Referrals, and Care Coordination.

Given the known impacts of social, behavioral, and economic factors on health and longevity, health policy reforms are being developed and deployed at both the state and federal levels that support the integration and alignment of health care and social services.

Reversal of Lead-Induced Acute Toxicity by Lipoic Acid with Nutritional Supplements in Male Wistar Rats.

Lead (Pb) is a pleiotropic toxicant. The potential role of oxidative stress injury that is associated with Pb poisoning suggests that antioxidants may enhance the efficacy of treatment designed to mitigate Pb-induced toxicity. The aim of this study is to investigate the comparative ameliorative potential of lipoic acid (LA) alone or in combination with calcium (Ca) and zinc (Zn). Pb acetate (50 mg/kg, intraperitoneally) was administered for 3 d. After 24 h of the last toxicant dose, LA (100 mg/kg, orally [po]) alone or in conjuction with Ca (50 mg/kg, po) and Zn (10 mg/kg, po) was administered for 3 d. Significant alterations in the concentration of urea, uric acid, triglycerides, cholesterol, aspartate amino transferase, alanine amino transferase, lipid peroxidation, and reduced glutathione as well as alterations in enzyme activity of δ-aminolevulinic acid (ALA) dehydratase were observed following acute Pb exposure. These findings were also supported by elevated mean DNA damage and Pb body burden in blood and soft tissues compared to controls (p ≤ 0.05). Three d posttreatment with LA along with Zn and Ca could significantly restore the biochemical parameters and Pb body burden to near-normal status through antioxidant activity or by preventing bioaccumulation of Pb within the blood and tissues of experimental rats.

Effect of NaCl stress on chlorophyll biosynthesis, proline, lipid peroxidation and antioxidative enzymes in leaves of salt-tolerant and salt-sensitive barley cultivars

In this research, the salt tolerance of salt-tolerant (Avci-2002) and salt-sensitive (Tokak 157/37) barley cultivars was analyzed. The seedlings were grown in a hydroponic culture containing 0, 100, 200 and 300 mM NaCl. The decrease in dry weight was more significant in Tokak 157/37 than in Avci-2002. On the other hand, no significant correlations were found between the altered d ­aminolevulinic acid dehydratase (ALAD) activity and the chlorophyll content. The chlorophyll and carotenoid contents in leaves of Avci-2002 decreased significantly at only 300 mM NaCl, whereas the pigment contents of Tokak 157/37 decreased at all NaCl concentrations. The increase in proline content was significantly higher in Avci-2002 than in Tokak 157/37 at 300 mM NaCl. The lipid peroxidation level measured in terms of malondialdehyde (MDA) content was higher in Tokak 157/37 than in Avci-2002. Salt stress caused significant increases in superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) activity. However, Avci-2002 had relatively lower SOD and CAT activities and higher POD and APX activities than Tokak 157/37. As a result, dry weight, pigments content, proline content, lipid peroxidation and activities of POD and APX results are in good correlation with supporting Avci-2002 being relatively salt-tolerant.

Evaluation of pesticide-induced oxidative stress from a gene–environment interaction perspective

Previously we reported that intensive agriculture workers exposed to pesticides had decreased levels of the intraerythrocyte enzymes delta-9-aminolevulinic acid dehydratase (ALA-D) and superoxide dismutase (SOD), very likely as a result of pesticide-induced oxidative stress. We have now examined in this population potential gene–environment interactions by modeling generalized estimating equations (GEE) adjusted for age, sex, body mass index and tobacco and alcohol consumption. Particularly, we assessed the interaction effects between plasma and erythrocyte cholinesterases (BChE and AChE, used as proxies for short- and long-term pesticide exposure, respectively) and a number of genetic polymorphisms of pesticide metabolizing enzymes such as paraoxonase-1 (PON1), glutathione-S-transferases (GST) and plasma cholinesterase variants (BCHE) on levels of erythrocyte antioxidant enzymes (SOD, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase and ALA-D). We observed significant interaction effects between BChE activity and PON1192R allele on catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities. BChE also interacted significantly with GSM1 null genotype on ALA-D and SOD. Regarding long-term pesticide exposure, a significant interaction was found between AChE and genotypes PON1192QR and PON1108CC on GR; between AChE and PON1192RR on SOD, and between AChE and GSTM1, GSTT1 and unusual BCHE variants on catalase activity. These findings suggest relevant gene–pesticide interactions and highlight the potential role of genetic risk factors in the pathomechanism of oxidative stress-induced degenerative diseases following pesticide exposure.

The effects of resistance exercise and post‐exercise meal timing on the iron status in iron deficient rats

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin (Hb) in iron deficient rats. Post‐exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post‐exercise nutrition timing on Hb synthesis are unclear. This study investigated the effect of post‐exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ‐aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the Hb concentration in iron deficient rats. Male 4‐week‐old Sprague‐Dawley rats were fed an iron deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/d, 3 d/w) for 3 weeks. The rats were divided into a group fed a post‐exercise meal early after exercise (E) or a group fed the meal 4h after exercise (L). A single bout of exercise after resistance exercise training for 3 weeks increased the bone marrow ALAD activity, plasma iron concentration and transferrin saturation (TfS). Although the plasma iron concentration and TfS were lower in the E group than in the L group after a single bout of exercise, the basal hematocrit, Hb and total iron binding capacity after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post‐exercise meal timing has no effect on the Hb concentration in iron deficient rats.

A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality

A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

Oral administration of diphenyl diselenide potentiates hepatotoxicity induced by carbon tetrachloride in rats

Carbon tetrachloride (CCl4) is a model for studying free radical-induced liver injury and screening hepato-protective drugs. Numerous studies have reported the involvement of oxidative stress in CCl4-induced liver damage and the hepato-protective effects mediated by different antioxidants. The present study examined the effects of diphenyl diselenide, (PhSe)2, on hepatotoxicity induced by CCl4 in rats. To this end, male Wistar rats received (PhSe)2 by oral route at the dosage of 31.2 mg/kg for one or two days. After the second day of treatment, rats received CCl4 orally in a single dose. The liver and kidney were utilized for determination of histopathology, biochemical [aspartate (ALT) and alanine (AST) aminotransferases, alkaline phosphatase (ALP), total bilirrubin (TB) and gamaglutamyl transferase (GGT)] and toxicological parameters [thiobarbituric reactive species (TBARS) levels, catalase activity, ascorbic acid, nonprotein thiols (NPSH) and aminolevulinate dehydratase (-ALA-D) activity]. Repeated administration of (PhSe)2 caused a marked potentiation of hepatotoxicity induced by CCl4 exposure, as manifested by an increase in biochemical parameters (AST, ALT, ALP, GGT and BT) and severe alteration in histopathology. This study also demonstrated a potentiation of TBARS levels and a consequent depletion of important antioxidant defenses including catalase and ascorbic acid. Pre-treatment with a single dose of (PhSe)2 prevented the effect of strychnine, a substrate for CYPs, abolishing lethality in mice. This result indicates that (PhSe)2 prevented animal death, suggesting an activator action of (PhSe)2 in CYPs. This study clearly indicates that (PhSe)2 potentiated acute hepatic damage induced by CCl4.

Delta‐Aminolevulinic Acid Dehydratase (ALAD) polymorphism that modulates lead toxicity is increased among autistic children

Chronic low dose exposure to lead in the environment has been associated with impaired cognitive development in children and adolescents. The risk of toxicity with low level lead exposure is likely to be determined, in part, by individual genetic susceptibility. The ALAD G177C polymorphism yields two co-dominant alleles, ALAD-1 and ALAD-2, which have been implicated in susceptibility to lead toxicity. The ALAD 1-2/2-2 variants have been associated with higher lead levels in blood and tissues and may lead to oxidative damage. The frequency of ALAD1 and ALAD2 genotypes was evaluated in 400 autistic children and 247 controls using ABI PRISM 7700 sequence detection system and allele-specific primer-probe sets from ABI Assays by Design. Plasma levels of glutathione and the GSH/GSSG ratio were quantified using HPLC with electrochemical detection. The frequency of ALAD 1-2/2-2 was 20.9% among cases and 13.8% among controls with an odds ratio of 1.66 (95% CI: 1.06, 2.62; p<0.02). In addition, significant gene-gene interactions were observed between ALAD 1-2/2-2 and the reduced folate carrier (RFC1 A80G), transcobalamin II (TCN2 C776G) and paroxonase 1 (PON1 T172A) genotypes. Glutathione levels and the GSH/GSSG ratio were lower among the ALAD 1-2/2-2 autistic children. These results suggest that the frequency of ALAD 1-2/2-2 genotype is increased among autistic children and may further exacerbate oxidative stress.

Protective effects of fruit extracts of Hippophae rhamnoides L. against arsenic toxicity in Swiss albino mice

Seabuckthorn (Hippophae rhamnoides L.) berry has a long history of applications as a food and medicinal ingredient in eastern countries. The present study was carried out to investigate the effect of different fruit extracts of H. rhamnoides on altered biochemical parameters indicative of haematological alterations, tissue oxidative stress, and arsenic concentration in arsenic-exposed mice (2.5 mg/kg body weight, intraperitoneally). Two aqueous extracts (at room temperature and under reflux condition) and an ethanolic extract of H. rhamnoides at a dose of 500 mg/kg body weight were co-administered daily during arsenic exposure in mice for 3 weeks. Exposure to arsenic led to a significant inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity, suggesting disturbed haem synthesis pathway. Arsenic also caused significant depletion of reduced hepatic glutathione (GSH) level, glutathione S-transferase (GST) and glutathione peroxidase and catalase activities, while it increased the level of thiobarbituric acid reactive substance (TBARS), suggesting liver oxidative stress. Most of the altered biochemical variables responded favorably to the co-supplementation of H. rhamnoides, particularly the aqueous fruit extract, extracted at room temperature (HF-WRT). However, arsenic concentration in blood and tissues remained unchanged, suggesting the lack of chelating property of fruit extract of H. rhamnoides. The present study, thus, led us to conclude that the fruit extract of H. rhamnoides has a significant protective role against arsenic-induced oxidative injury. However, it lacks the ability to remove arsenic from the binding sites, suggesting that the herbal extract could be co-administered with a chelating agent of known efficacy during treatment of arsenic to achieve the optimum effect of chelation treatment.

The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism.

From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propionic acid is metabolized via the methylcitric acid pathway. The genes encoding enzymes of this pathway are organized in a cluster in the order prpR, prpB, prpC, acnM, ORF5 and prpD, with prpR transcribed divergently from the other genes. (i) prpC encodes a 2-methylcitric acid synthase (42720 Da) as shown by the measurement of the respective enzyme activity, complementation of a prpC mutant of Salmonella enterica serovar Typhimurium and high sequence similarity. (ii) For the translational product of acnM the function of a 2-methyl-cis-aconitic acid hydratase (94726 Da) is proposed. This protein and also the ORF5 translational product are essential for growth on propionic acid, as revealed by the propionic-acid-negative phenotype of Tn5-insertion mutants, and are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid as shown by the accumulation of the latter, which could be purified as its calcium salt from the supernatants of these mutants. In contrast, inactivation of prpD did not block the ability of the cell to use propionic acid as carbon and energy source, as shown by the propionic acid phenotype of a null-allele mutant. It is therefore unlikely that prpD from R. eutropha encodes a 2-methyl-cis-aconitic acid dehydratase as proposed recently for the homologous prpD gene from S. enterica. (iii) The translational product of prpB encodes 2-methylisocitric acid lyase (32314 Da) as revealed by measurement of the respective enzyme activity and by demonstrating accumulation of methylisocitric acid in the supernatant of a prpB null-allele mutant. (iv) The expression of prpC and probably also of the other enzymes is regulated and is induced during cultivation on propionic acid or levulinic acid. The putative translational product of prpR (70895 Da) exhibited high similarities to PrpR of Escherichia coli and S. enterica, and might represent a transcriptional activator of the sigma-54 family involved in the regulation of the other prp genes. Since the prp locus of R. eutropha was very different from those of E. coli and S. enterica, an extensive comparison of prp loci available from databases and literature was done, revealing two different classes of prp loci.

Cutaneous porphyria in a neonate with tyrosinaemia type 1

A term infant born to consanguineous parents presented at birth with hypoglycaemia, thrombocytopenia, coagulopathy and hyperbilirubinaemia associated with polycythaemia due to delayed cord clamping. Despite phototherapy and correction of polycythaemia by partial exchange transfusion, coagulopathy, hypoglycaemia and conjugated hyperbilirubinaemia persisted, suggesting hepatic failure. Metabolic work-up led to the diagnosis of tyrosinaemia type 1 on day 4. Two--(2-nitro-4-trifluoromethylbenzoyl)--1,3 cyclohexanedione (NTBC) treatment, started on day 5, resulted in progressive clinical improvement and unambiguous biochemical response. Severe skin purpuric lesions occurred in areas exposed to phototherapy. These resolved slowly after its discontinuation. Urine analysis sampled just before and 6 days after starting NTBC treatment showed high levels of type 1 coproporphyrin isomers. Such findings do not seem directly related to tyrosinaemia type 1 where succinylacetone inhibits delta-aminolevulinic acid (delta-ALA) dehydratase and where the accumulation of delta-ALA results in neurotoxicity without photosensitivity. We describe a cutaneous form of porphyria in a neonate presenting with severe liver failure due to tyrosinaemia type 1. This porphyria is tentatively attributed to a secondary accumulation of coproporphyrins due to cholestasis, as reported in the bronze baby syndrome and recently described in neonates with purpuric phototherapy-induced eruption, rather than to a primary defect of porphyrin metabolism. The hypothesis of a direct effect of tyrosinaemia type 1 on porphyrin excretion is also discussed.

Selected trace elements and organochlorines: Some findings in blood and eggs of nesting common eiders (Somateria mollissima) from Finland

AbstractIn 1997 and 1998, we collected blood samples from nesting adult female commoneiders (Somateria mollissima) at five locations in the Baltic Sea near coastal Finland and analyzed them for lead, selenium, mercury, and arsenic. Eggs were collected from three locations in 1997 for analysis of selenium, mercury, arsenic, and 17 organochlorines (OCs). Mean blood lead concentrations varied by location and year and ranged from 0.02 ppm (residues in blood on wet weight basis) to 0.12 ppm, although one bird had 14.2 ppm lead in its blood. Lead residues in the blood of eiders were positively correlated with the stage of incubation, and lead inhibited the activity of the enzyme delta‐aminolevulinic acid dehydratase (ALAD) in the blood. Selenium concentrations in eider blood varied by location, with means of 1.26 to 2.86 ppm. Median residues of selenium and mercury in eider eggs were 0.55 and 0.10 ppm (residues in eggs on fresh weight basis), respectively, and concentrations of both selenium and mercury in eggs were correlated with those in blood. Median concentrations of p,p′‐dichlorodiphenyldichloroethylene in eggs ranged from 13.1 to 29.6 ppb, but all other OCs were below detection limits. The residues of contaminants that we found in eggs were below concentrations generally considered to affect avian reproduction. The negative correlation of ALAD activity with blood lead concentrations is evidence of an adverse physiological effect of lead exposure in this population.

Biomarkers and fluctuating asymmetry as indicators of pollution‐induced stress in two hole‐nesting passerines

Abstract1. The stress caused by air pollutants was studied at biochemical, morphological and ecological levels in the Pied Flycatcher, Ficedula hypoleuca, and Great Tit, Parus major, nestlings in 10 study sites along the pollution gradient of a copper smelter.2. First, stress was measured using four biomarkers from blood and liver: ethoxyresorufin O‐deethylase (EROD) enzyme, haemoglobin, stress protein Hsp70 and delta‐aminolevulinic acid dehydratase (ALA‐d) enzyme. Second, the amount of fluctuating asymmetry (FA) in the length of the 3rd primary and the outermost rectrix (in P. major) and the length and thickness of the tarsus were measured. These stress indicators were further examined in relation to the breeding performance of birds, i.e. the ecological response.3. EROD activity was increased in F. hypoleuca nestlings near the pollution source. In P. major, it was not related to the pollution gradient but correlated well with the proportion of starved nestlings, i.e. the nutritional stress during the nestling period. The variations in haemoglobin, Hsp70 and ALA‐d enzyme activity were not significantly related to pollution gradient.4. The tarsus length of F. hypoleuca nestlings and primary length of P. major nestlings showed increased asymmetry in the vicinity of the pollution source. Breeding success decreased towards the pollution source in both species.5. In F. hypoleuca, the pollution‐related stress was verified at all three target levels. In P. major, pollution‐related effects were found at morphological and ecological levels but not at the biochemical level, which suggests that direct toxic effect of heavy metals was not the main mechanism in this species.

Biomarker Assessment of the Effects of Coal Strip-Mine Contamination on Channel Catfish

A suite of biomarkers was used to evaluate acute (1-day) to semichronic (3-month) heavy metal-induced toxicity in channel catfish,lctalurus punctatus,caged at an abandoned strip mine and a noncontaminated reference site. Assays performed include indicators of metabolic, hematological, osmoregulatory, and genotoxic stress. Two cage designs were used to evaluate the importance of exposure routes: one excluding contact with the sediments and the other allowing contact with water and sediments. Significant DNA strand breakage was observed in catfish exposed to both exposure regimens, but evidence of DNA repair was observed only in water-exposed catfish. Transient increases in hemoglobin,δ-aminolevulinic acid dehydratase, and hematocrit levels were observed at 1 month's exposure for both exposure regimens, followed by a return to control levels for the duration of the study. Environmental conditions (i.e., weather-related changes in water quality) may have contributed to the variable plasma chloride and glucose levels observed in all catfish exposed to strip-mine wastes. The transient changes in biomarkers followed by a return to reference values represent an initial stress and an acclimation to normal levels.

ChemInform Abstract: Synthesis of Analogues of 5‐Aminolaevulinic Acid and Inhibition of 5‐Aminolaevulinic Acid Dehydratase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Relation between Polymorphism of δ‐Aminolevulinic Acid Dehydratase and Some Parameters in Lead Workers

Relation between Polymorphism of δ‐Aminolevulinic Acid Dehydratase and Some Parameters in Lead Workers

Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves

Cesium chloride (CsCl) treatment of greening primary leaves of barley for 8 h inhibited chlorophyl] accumulation in a concentration‐dependent manner and led to the accumulation of excessive amounts of uroporphyrin(ogen) III (URO[gen]) and to a minor extent of heptacarboxylporphyrin(ogen). When dark‐grown leaves were incubated with CsCl, accumulation of URO(gen) was observed only after feeding of the tetrapyrrole precursor 5‐aminolevulinic acid. Western blot analysis showed no apparent difference in content of uroporphyrinogen decarboxylase (EC 4.1.1.37, UROD) or selected proteins involved in tetrapyrrole biosynthesis in extracts of CsCl‐incubated (15 mM) versus control leaves. UROD activity was drastically decreased upon CsCl treatment in leaves incubated in the dark or in the light (44 and 86%, respectively). Selected preceding enzymes of the tetrapyrrole biosynthetic pathway, 5‐aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) and porphobilinogen deaminase (EC 4.3.1.8, PBGD), were influenced only to a minor extent under standard incubation conditions (15 mM CsCl). Furthermore, the ALA synthesizing capacity did not differ in leaves incubated with and without Cs− cations. UROD activity of crude homogenates from control plants and after partial purification was reduced to 56 and 80%, respectively, upon addition of 10 mM CsCl. Equal concentrations of KCl were not inhibitory. Enzyme assays of the same barley extract in the presence of CsCl yielded no effect on ALAD and a minor loss of PBGD activity. The initial visible cytotoxic effect of CsCl appeared to be a selective inhibition of UROD resulting in accumulation of photosensitizing URO (gen). Consequences of the diminished UROD activity on early steps of the tetrapyrrole biosynthesis and its functional and regulatory significance for the porphyrin synthesis are discussed.

Inhibition of 5-aminolevulinic acid (ALA) dehydratase by undissociated levulinic acid during ALA extracellular formation by Rhodobacter sphaeroides

The inhibition of ALA dehydratase by levulinic acid during ALA extracellular formation of Rhodobacter sphaeroides correlated with the concentration of undissociated form of levulinic acid irrespective of culture pH. The inhibition constant, Ki, of intracellular ALA dehydratase by Dixon plots was 2.95 μM. Undissociated levulinic acid therefore functions as an inhibitor of ALA dehydratase.