Effect of lead and mercury on chlorophyll synthesis in mung bean seedlings

Germinating seeds of mung bean were treated with different concentrations of lead and mercury. Estimations of δ-aminolevulinic acid dehydratase activity, chlorophyll and protein contents were performed. The metals were found to inhibit δ-aminolevulinic acid dehydratase activity and decrease total chlorophyll content, suggesting the possible regulatory role of the enzyme in chlorophyll synthesis. The δ-aminolevulinic acid dehydratase activity was localized exclusively in chloroplasts.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Renal enzyme changes in pekin ducks (Anas platyrynchos) after combined administration of methylmercury,cadmium and lead

1. The combined effect of methylmercury, cadmium and lead on renal enzymes of pekin ducks was studied.2. Renal acid phosphatase and glutathione S-transferase activities were not affected by heavy metal treatment.3. Renal δ-aminolevulinic acid dehydratase activity was decreased significantly in ducks treated with lead alone or when lead was co-administered with methylmercury.4. Renal cytochrome c oxidase activity was decreased significantly when methylmercury was co-administered with cadmium and/or lead.5. The findings suggest that lead had the main effect on 5-aminolevulinic acid dehydratase and methylmercury had the main effect on cytochrome c oxidase activity. Interaction effect was also observed in cytoehrome c oxidase activity.     

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Nonproliferating cultures of adult rat hepatocytes were found to lose 60–70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (95%) in the activity of the zinc metalloenzyme, δ-aminolevulinic acid dehydratase, as well as a loss (>85%) in the cellular content of immunoreactive δ-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both δ-aminolevulinic acid dehydratase activity and immunoreactive protein. Since the spontaneous, selective loss of cellular zinc appears to have specific effects on a relevant hepatic function, this culture system constitutes a novel $\text{in}$$\text{vitro}$ model of zinc deficiency in mature liver.     

Effect of EMD-IT-5914 on Chlorophyll Synthesis in Leaves of Pennisetum typhoides Seedlings

EMD-IT-5914 (5-dimethylamino-methylene-2-oxo-4-phenyl-2,5-dihydrofurane-carbonitril-(3)) inhibited chlorophyll a formation almost completely and chlorophyll b and total carotenoids up to 80% of the control, but did not appreciably affect the activity of the enzyme system succinyl-CoA synthetase/δ-aminolevulinic acid synthetase. The activity of δ-aminolevulinic acid dehydratase was not found limiting. In contrast, the herbicide strongly inhibited the activity of porphobilinogenase, and the reaction kinetics pointed towards a non-competitive type of inhibition. The results are discussed in relation to the possible role of EMD-IT-5914 in chlorophyll biosynthesis.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of Nicotiana tabacum

The activity of a sequence of enzymes involved in chlorophyll biosynthesis (δ-aminolevulinic acid synthetase (ALAS), δ-aminolevulinic acid dehydratase, porphobilinogenase and chlorophyllase) was followed during greening of tobacco cell cultures under the influence of chloramphenicol (CAP). The photosynthetic enzymes ribulose diphosphate carboxylase (RuDPCO) and NADP linked glyceraldehyde dehydrogenase (NADP-GDH) were used as markers for penetration and action of the inhibitor. RuCPCO was inhibited at concentrations of CAP which still allowed good chlorophyll accumulation. The enzymes of chlorophyll biosynthesis, the activity of which increased during illumination and CAP treatment, behaved like NADP-GDH which is known to be synthesized in the cytoplasm. The results suggest that synthesis of enzymes of chlorophyll biosynthesis takes place in the cytoplasm. Decreasing light induced increment of ALAS activity caused by CAP may possibly be taken as an indication that things are more complicated with this enzyme.     

An immunological study of δ-aminolevulinic acid dehydratase specificity consistent with the phylogeny of species

Rabbit antibody directed to homogeneously purified mouse liver δ-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of δ-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined.     

The Mg insertion step in chlorophyll biosynthesis.

A developing chloroplast preparation obtained from greening cucumber cotyledons is able to bring about the synthesis of Mg-protoporphyrin-IX and/or Mg-protoporphyrin-IX monomethyl ester. l-glutamate, δ-aminolevulinic acid, and protoporphyrin-IX can serve as precursors for Mg-protoporphyrin synthesis. However, when δ-aminolevulinic acid or protoporpyrin are used, no Mg-protoporphyrin is formed unless l-glutamate is also added. Mg-Protoporphyrin synthesis with δ-aminolevulinic acid plus l-glutamate, or proto-porphyrin plus l-glutamate, is much more active than with l-glutamate alone. Therefore, it is apparent that l-glutamate plays a role in the Mg chelation step in chloroplasts. α-Keto-glutarate can replace l-glutamate in this role; glutamine cannot. ATP is also required for Mg chelation. The role of l-glutamate in the Mg insertion step is not yet understood, except that l-glutamate itself does not need to be converted to porphyrins in this process, because Mg-protoporphyrin can be synthesized from protoporphyrin and l-glutamate even in the presence of the δ-aminolevulinic acid dehydratase inhibitor, levulinate.     

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

Porphyrin metabolism in lead and mercury treated bajra (Pennisetum typhoideum) seedlings

Lead and mercury inhibited porphyrin biosynthesis significantly in the germinating seeds of bajra (Pennisetum typhoideum). Both 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were inhibited by these metals. A comparative study of the inhibition of these two enzymes under invivo andin vitro conditions showed that 5-aminolevulinic acid dehydratase is the major site of action of heavy metals in porphyrin biosynthesis. Further, over-all production of porphyrinsviz., protoporphyrin-IX, Mg-protoporphyrin (ester) and protochlorophyllide was repressed by lead and mercury in both light and dark grown seedlings. Similarly, chlorophylla and chlorophyllb and total chlorophyll contents in dark-grown seedlings were also significantly decreased, suggesting the impairment of chlorophyll biosynthesis by lead and mercury in germinating seedlings.     

Automated determination of delta-aminolevulinic acid dehydratase activity in human erythrocytes

An automated assay for the estimation of δ-aminolevulinic acid dehydratase activity in human erythrocytes has been devised. The present design allows the performance of thirteen analyses per hour. Samples are hemolysed by saponin in the flow system. Lateral interaction between consecutive samples is negligible and repeatability of analysis is excellent. The analytical values of the automated and manual procedures correlate strongly. In randomly selected, hospitalized patients the mean value of δ-aminolevulinic acid dehydratase activity is significantly lower than that found in a normal population.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.     

Characterization of a cDNA encoding 5-aminolevulinic acid dehydratase in tomato (Lycopersicon esculentum Mill.)

Summary A full-length cDNA clone encoding tomato (Lycopersicon esculentum Mill.) 5-aminolevulinic acid dehydratase (ALAD) was isolated and characterized. The primary structure predicts a 430-amino acid precursor which comprises a 41.7 kDa, 388-amino acid mature protein and a 47-amino acid transit sequence. The tomato primary sequence shows extensive homology to those of pea and spinach. Southern analysis indicated that 1 to 2 copies of the ALAD gene are present in the tomato genome. Northern blot analysis shows differential expression in various tomato organs, and constitutive developmental expression in tomato fruits.Abbreviations ALA 5-aminolevulinic acid - ALAD 5-aminolevulinic acid dehydratase (EC 4.2.1.24)     

Exposure to Low Level of Arsenic and Lead in Drinking Water from Antofagasta City Induces Gender Differences in Glucose Homeostasis in Rats

Populations chronically exposed to arsenic in drinking water often have increased prevalence of diabetes mellitus. The purpose of this study was to compare the glucose homeostasis of male and female rats exposed to low levels of heavy metals in drinking water. Treated groups were Sprague-Dawley male and female rats exposed to drinking water from Antofagasta city, with total arsenic of 30 ppb and lead of 53 ppb for 3 months; control groups were exposed to purified water by reverse osmosis. The two treated groups in both males and females showed arsenic and lead in the hair of rats. The δ-aminolevulinic acid dehydratase was used as a sensitive biomarker of arsenic toxicity and lead. The activity of δ-aminolevulinic acid dehydratase was reduced only in treated male rats, compared to the control group. Treated males showed a significantly sustained increase in blood glucose and plasma insulin levels during oral glucose tolerance test compared to control group. The oral glucose tolerance test and the homeostasis model assessment of insulin resistance demonstrated that male rats were insulin resistant, and females remained sensitive to insulin after treatment. The total cholesterol and LDL cholesterol increased in treated male rats vs. the control, and triglyceride increased in treated female rats vs. the control. The activity of intestinal Na+/glucose cotransporter in male rats increased compared to female rats, suggesting a significant increase in intestinal glucose absorption. The findings indicate that exposure to low levels of arsenic and lead in drinking water could cause gender differences in insulin resistance.     

Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme content and delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10^?7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.