Descripción: 1. 1. δ-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activity reached its maximum when callus were 11 days old. 2. 2. The presence of a compound which seems to control δ-aminolaevulinate synthetase activity was demonstrated. The enzyme was present in the soluble fraction and was very labile. 3. 3. When crude extracts or 500 × g supernatant were stored at 4-6°, the apparent activity of δ-aminolaevulinate synthetase increased by as much as 3-6 times, while the activities of δ-aminolaevulinate dehydratase and succinyl-CoA synthetase did not significantly change during the storage. Activation was dependent on concentrations of cells suspensions during disruption and aging. 4. 4. Gel filtration with Sephadex G-25 of 2000 × g supernatants produced an enzyme fraction 30% more active. An increase in enzyme activity was observed when dark-grown callus were exposed to light. 5. 5. The addition of ATP, gibberellic acid and δ-aminolaevulinate to the culture media diminished activity; iron deficiency also produced an δ-aminolaevulinate synthetase less active. © 1971.

Descripción: 1. 1. The reaction between 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the SH groups of soybean callus succinyl-CoA synthetase has been investigated. At pH 8-6 and 25° C, four SH groups are titrable with DTNB in the native enzyme. No additional thiol groups have been revealed after unfolding of the protein with 8 M urea. 2. 2. The loss of enzymatic activity paralleled the decrease in the number of free SH groups. As with p-mercuribenzoate and other mercurials, reaction with DTNB also resulted in dissociation of the enzyme into subunits. © 1974.

Descripción: 1. 1. The activity of Succinyl CoA Synthetase (Suc CoA-S), Cysthationase, Rhodanese, Aminolevulinate Synthetase (ALA-S) and Aminolevulinate Dehydratase (ALA-D) was studied in old (405-407 subcultures) and young (34-36 subcultures) soybean callus clones as a function of the days of growing. 2. 2. Suc CoA-S, ALA-S and ALA-D activities were much lower in old than in young callus, while the activity of Cysthationase and Rhodanese was higher in old callus. 3. 3. ALA-S reached its maximum activity when Rhodanese and Cysthationase their minimum, on the 11th day of growth. It is suggested that the cellular content of a possible thio-compound which would regulate ALA-S activity, is controlled through its degradation by Rhodanese. © 1980.

Descripción: 1. 1. The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in endosymbiote-free and endosymbiote-containing Crithidia deanei grown in a chemically defined medium: succinyl Coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), por- phobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. 2. 2. ALA and PBG were detected in C. deanei. The levels of free porphyrins was low. Heme concentration was nil. 3. 3. The activity of ALA-D. deaminase and PBGase was not detected in C deanei. 4. 4. The activity of Suc.CoA-S and ALA-S were twice higher in symbiote-containing than in aposymbiotic C. deanei. Aposymbiotic cells had a higher activity of DOVA-T than symbiote-containing cells. 5. 5. The level of Heme-S, measured using protoporphyrin as substrate, was twice as high in symbiotecontaining than in symbiote-free cells,. © 1985.

Descripción: 1. 1. Effects of various factors on chlorophyll, porphyrin and protein content, growth and on the activities of the enzymes involved in the earlier stages of tetrapyrrole synthesis, in cultured soybean cells, were studied. 2. 2. When dark-grown callus was exposed to light, it was found that the amount of porphyrins formed was not altered, but chlorophyll content as well as Succinyl CoA Synthetase (Suc.CoA-S), Aminolevulic Acid Synthetase (ALA-S), Aminolevulic Acid Dehydratase (ALA-D) and Porphobilinogenase (PBGase) activities increased. 3. 3. Addition of Aminolevulic Acid (ALA) to the medium culture, was found to stimulate porphyrin accumulation and to prevent growth; however chlorophyll content was not significantly modified. ALA-S was inhibited while both ALA-D and PBGase activities were enhanced. The action of puromycin and mitomycin added along with ALA to the media, was also studied, but neither of these inhibitors modified much the effects produced by ALA. 4. 4. Addition of Porphobilinogen (PBG), showed accumulation of uroporphyrin in the tissue; except inhibition of ALA-S, enzymes activities, protein and chlorophyll content were not modified. Evidence obtained would indicate that callus tissue was not permeable to PBG. 5. 5. Omission of iron from the culture medium, produced porphyrin accumulation and prevented growth. It has been consistently found that, the higher the content of porphyrins, the less the callus growth. Coproporphyrin was the major component of the porphyrins formed in ALA supplemented or iron deficient media. ALA-S and ALA-D were reduced under iron deficiency. 6. 6. The addition of ATP to the media, did not affect porphyrin, protein, and chlorophyll synthesis, growth or ALA-D, but Suc.CoA-S, ALA-S and PBGase activities diminished. 7. 7. Gibberelic acid produced a measurable increase of PBGase, while diminished Suc.CoA-S and ALA-S. 8. 8. Succinate increased growth and inhibited ALA-S and ALA-D. 9. 9. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP), added to the medium produced accumulation of porphyrins, consequently, ALA-S was greatly inhibited and growth prevented. PBGase was also diminished. 10. 10. Coproporphyrinogenase and Decarboxylases activities were hardly detected in most experiments, and are limiting. 11. 11. The complex pattern of results obtained is discussed. © 1975.

Descripción: The high levels of δ-aminolevulinate synthetase (ALA-S) in Rhodopseudomonas palustris cells grown anaerobically in the light (Ph) decrease to those found in cells grown aerobically in the dark (A), when the former cultures were vigorously oxygenated; simultaneously bacteriochlorophyll (Bchl) synthesis abruptly halted leading to diminished steady-state specific Bchl content. When flushing oxygen was interrupted, enzymic activity increased, whether chloramphenicol was present or not in the medium; if the protein synthesis inhibitor was added when oxygenation started, ALA-S declined in the same fashion as in its absence, but thereafter reactivation of the enzyme was lower than before. Succinyl-CoA-synthetase and ALA-dehydratase activities were also measured under the conditions described, and no changes at all have been observed. The existence of different forms of ALA-S in R. palustris depending on growth conditions is postulated along with the formation of low molecular weight factors which can modulate ALA-S activity by binding to the enzyme; a widespread mechanism in the adaptation of micro-organisms to changes in environment. It is also proposed that this particular regulatory phenomenon, could be referred to as a switch off/on mechanism controlling ALA-S activity in R. palustris. © 1987.

Descripción: An enzyme, aminolaevulinate dehydratase (5-aminolaevulinate hydro-lyase (adding 5-aminolaevulinate and cyclizing), EC 4.2.1.24), which catalyzes the following reaction: {A figure is presented} has been isolated from yeast. Its purification and some of its properties have been studied. 5-Aminolaevulinate synthetase, another enzyme involved in porphyrin biosynthesis, has been detected in the mitochondrial particles. © 1967.

Descripción: 1. 1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of δ-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver. © 1990.

Descripción: Erythropoietic Protoporphyria (EPP) is an inherited deficiency of ferrochelatase, the last enzyme of the heme pathway. Under general anaesthesia, some patients develop neurological dysfunction suggesting upregulation in heme biosynthesis similar to that described for acute porphyrias after xenobiotic administration. Our aim has been to evaluate whether Isoflurane induces alterations in the heme pathway in a mouse model for EPP. Administration of Isoflurane (a single dose of 2 ml/kg, i.p) to wild-type (+/+), heterozygous (+/Fechm1Pas) and homozygous (Fechm1Pas/Fech m1Pas) mice, was evaluated by measuring the activity of δ-Aminolevulinic acid synthetase (ALA-S) and Porphobilinogen-deaminase (PBG-D) in different tissues, as well as Heme oxygenase (HO), cytochrome P-450, CYP2E1 and glutathione levels in liver. Porphyrin precursors were measured in 24h-urine samples. Fechm1Pas/Fechm1Pas mice receiving anaesthesia show enhanced ALA-S and CYP2E1 activities in the liver and increased urinary excretion of porphyrin precursors. No alterations were found in either PBG-D or HO activities. Diminished glutathione levels suggest that anaesthesia may produce oxidative stress in these animals. In conclusion, Isoflurane induces ALA-S activity and increased excretion of porphyrin precursors in EPP mice. These findings appear to confirm our previous hypothesis and indicate that Isoflurane may be an unsafe anaesthetic not only for patients with acute porphyrias but also for individuals with non acute porphyrias. Copyright © 2009 C.M.B. Edition.

Descripción: 1. 1. The porphyrinogenic ability of several antineoplasics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of δ-aminolaevulinate synthase(ALA-S). were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan. notwithstancling the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo. © 1988.

Descripción: Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain mince, with [1,23H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 μM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.

Descripción: The porphyrias are a group of inherited metabolic disorders of heme biosynthesis which result from a partial deficiency in one of its seven specific enzymes, after its first and rate limiting enzyme, delta-aminolevulinic acid synthetase. They can be classified on the basis of their clinical manifestations into cutaneous, acute and mixed disorders. Acute intermittent porphyria (AIP) is the most common type of hepatic acute porphyrias, inherited as an autosomal dominant trait, caused by a defect in the gene which codifies for the heme enzyme porphobilinogen deaminase. Its prevalence in the Argentinean population is about 1:125,000. A partial deficiency in another enzyme, protoporphyrinogen oxidase, produces variegate porphyria (VP), the second acute porphyria most frequent in the Argentinean population (1:600,000). Here, we review all the mutations we have found in 46 AIP and 9 VP unrelated Argentinean patients. To screen for mutations in symptomatic patients, we have proposed a geneticresearch strategy.

Descripción: 1. 1. The present work undertakes a comparative study on the hexachlorobenzene (HCB) porphyria induction in female rats of Wistar and CHBBTHOM strains. The purpose was to characterize the CHBBTHOM strain with respect to the haem metabolic pathway, its regulatory mechanisms and its response to foreign drugs. 2. 2. After 7 weeks of treatment it was observed that the hepatic porphyrins increased 140 times, ALA-synthase 4 times and PCL was 73% inhibited in the Wistar strain. 3. 3. On the other hand the animals of CHBBTHOM strain showed lesser alteration on these parameters; hepatic porphyrins increased only 3-fold, ALA-synthase 1.7-fold and PCL was only 22% inhibited. 4. 4. Total iron liver content was nearly equal in both strains of rats. 5. 5. The results obtained would indicate that the lower susceptibility of the CHBBTHOM strain to acquire porphyria does not seem to be due to either: (1) congenital alterations of any parameters of the haem metabolic pathway, since the behaviour of normal animals from both strains was similar; or (2) a lower hepatic iron content in such animals. 6. 6. These findings would suggest that the differential response to HCB to this strain would be looked for in another metabolic pathway, such as that involved in the metabolization process of the toxic. © 1989.

Descripción: 1. 1. No changes in ALA-S activity were observed when different preparations of R. palustris were stored at 4°C for various periods of time. 2. 2. Mixing supernatants from pigmented and decoloured R. palustris cells, showed that the activity of ALA-S was several times higher than expected, suggesting the presence of an activator. 3. 3. Supernatants from photosynthetically and aerobically grown cells were heated and the effect of the protein-free supernatant was tested on both red and white supernatants. The heated supernatant from aerobic cells increased ALA-S when added to red and white preparations, but the heated red supernatant only activated red supernatant and had no action on the white cells enzyme. 4. 4. By gel filtration on Sephadex G-25 of cell free extracts from R. palustris either aerobically or anaerobically grown, a low molecular weight compound was separated, which added back to the homologeous enzyme enhanced its activity confirming the existence of one or two low-molecular weight and heat-stable factors which would act stimulating ALA-S activity. 5. 5. A scheme is proposed to explain the role of these factors on the control of ALA-S in R. palustris. © 1980.

Descripción: Riboflavin, an essential cofactor for all organisms, is biosynthesized in plants, fungi and microorganisms. The penultimate step in the pathway is catalyzed by the enzyme lumazine synthase. One of the most distinctive characteristics of this enzyme is that it is found in different species in two different quaternary structures, pentameric and icosahedral, built from practically the same structural monomeric unit. In fact, the icosahedral structure is best described as a capsid of twelve pentamers. Despite this noticeable difference, the active sites are virtually identical in all structurally studied members. Furthermore, the main regions involved in the catalysis are located at the interface between adjacent subunits in the pentamer. Thus, the two quaternary forms of the enzyme must meet similar structural requirements to achieve their function, but, at the same time, they should differ in the sequence traits responsible for the different quaternary structures observed. Here, we present a combined analysis that includes sequence-structure and evolutionary studies to find the sequence determinants of the different quaternary assemblies of this enzyme. A data set containing 86 sequences of the lumazine synthase family was recovered by sequence similarity searches. Seven of them had resolved three-dimensional structures. A subsequent phylogenetic reconstruction by maximum parsimony (MP) allowed division of the total set into two clusters in accord with their quaternary structure. The comparison between the patterns of three-dimensional contacts derived from the known three-dimensional structures and variation in sequence conservation revealed a significant shift in structural constraints of certain positions. Also, to explore the changes in functional constraints between the two groups, site-specific evolutionary rate shifts were analyzed. We found that the positions involved in icosahedral contacts suffer a larger increase in constraints than the rest. We found eight sequence sites that would be the most important icosahedral sequence determinants. We discuss our results and compare them with previous work. These findings should contribute to refinement of the current structural data, to the design of assays that explore the role of these positions, to the structural characterization of new sequences, and to initiation of a study of the underlying evolutionary mechanisms.

Descripción: The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1.2 M the growth curves were similar to those of its wild-type control, at 1 .5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations. © 2006 SGM.

Descripción: Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5′-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3β or HNF3β plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3β is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3β can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3β binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Descripción: Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5′-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5′-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.