Formation Of Δ-aminolevulinic Acid Research Articles

Influence of Selenium Supplementation on δ-Aminolevulinic Acid Formation in Greening Maize Leaf Segments

Influence of Selenium Supplementation on δ-Aminolevulinic Acid Formation in Greening Maize Leaf Segments

The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU(TPR)) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLU(TPR) at 2.4-Å resolution. Three non-canonical TPR motifs of each FLU(TPR) form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU(TPR)-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.

Effect of arsenic on δ-aminolevulinic acid formation in greening maize leaf segments

Supply of 0.5 mM Na2AsO4 significantly decreased the δ-amino levulinic acid (ALA) content and total chlorophylls in greening maize leaf segments with the effect being more substantial for the later. Inclusion of precursors of chlorophyll also affected the ALA content. Thus, % decrease by arsenic (As) was significantly reduced in the presence of glutamine and MgCl2, but was increased by glycine and succinate. ALA formation and chlorophyll content were significantly decreased by 0.5 mM Na2AsO4 during levulinic acid treatment in light as well as dark with more severe effect in light. Difference in ALA accumulation in light and dark (L–D) was also substantially decreased by higher concentrations of As. Inclusion of succinate and MgCl2, respectively, increased and decreased the % inhibition of ALA formation in light. In dark the addition of 2-oxoglutaric acid (2-OG), succinate and glycine caused an increase in % inhibition of ALA formation, while glutamate caused a decrease in inhibition. Inclusion of adenosine tri phosphate (ATP) increased the ALA formation in light in presence of As only, while pyridoxal phosphate (PLP) and MgCl2 enhanced it under both conditions. The results demonstrate more prominent decrease in total chlorophylls than ALA content and/or formation in the presence of As, indicating the involvement of other steps of chlorophyll biosynthesis in addition to ALA synthesis. Substantial decrease under dark at higher concentrations of As and protective effect of cofactors, such as, MgCl2, PLP and ATP in ALA formation in light suggests that chloroplastic ALA synthesizing activity is more sensitive to the presence of As.

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

ObjectiveIn mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates. Design and methodsA systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at −80°C. ConclusionsA method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

Trafficking of heme and porphyrins in metazoa.

Trafficking of heme and porphyrins in metazoa.

The enantioselective participation of (S)- and (R)-diaminovaleric acids in the formation of δ-aminolevulinic acid in cyanobacteria

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.

Characterization of δ-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor delta-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1-[(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed.

Separation and partial characterization of enzymes catalyzing δ-aminolevulinic acid formation in Synechocystis sp. PCC 6803

Formation of the universal tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differential ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 μ m, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 μ m. GSA aminotransferase was inhibited 50% by 0.5 μ m gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 °C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.

Production of δ-aminolevulinate: Subcellular localization and purification of murine hepatic l-alanine: 4,5-dioxovaleric acid aminotransferase

1. 1. l-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C 57BL/6J and DBA/2J. 4. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of δ-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on l-alanine-AH-Sepharose. 8. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. 9. The yield of the purification was ca 90 μg of protein per gram liver wet weight. 10. 10. The purified enzyme had a subunit mol. wt of 146,000 ± 5000 as determined by electrophoresis under denaturing conditions.

The tRNA Required for in Vitro δ-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts

RNA is an essential component for the enzymic conversion of glutamate to delta-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA(Glu(UUC)), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA(Glu(UUC)) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA(Glu(UUC)) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [(3)H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA(Glu(UUC)) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA(Glu(UUC)) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA(Glu(UUC)) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [(3)H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.

Formation of δ-Aminolevulinic Acid from Glutamic Acid in Algal Extracts

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to delta-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg(2+), and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH(4))(2)SO(4) at 70% saturation. The proteins recovered from the (NH(4))(2)SO(4) precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH(4))(2)SO(4) precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography.

Stimulation of δ-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA

Formation of the chlorophyll and heme precursor delta-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond formation. Our results suggest that the RNA that is required for ALA formation may be functionally distinct from the glutamyl-tRNA species involved in protein synthesis.

RNA is required for enzymatic conversion of glutamate to δ-aminolevulinate by extracts of Chlorella vulgaris

Formation of δ-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell. extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNA glu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs With glutamate.

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.

Benzyladenine-induced Chlorophyll Formation in Etiolated Cucumber Cotyledons: Differential Responses of δ-aminolevulinic Acid Formation to Inhibitors of RNA ano Protein Synthesis in Darkness and then after Illumination

Summary The effect of benzyladenine (BA), applied alone or in combination with inhibitors of RNA and protein synthesis, on growth, chlorophyll and δ-aminolevulinic acid (ALA) accumulation in cucumber ( Cucumis sativus L.) cotyledons in darkness and then after illumination was studied. BA increased the rate of ALA accumulation in levulinic acid-treated etiolated cucumber cotyledons after 2–4 h in darkness. The results when inhibitors of RNA and protein synthesis were present suggested that BA in darkness leads to an acitivation of m-RNA that may already be present, but does not enhance the synthesis of new m-RNA. Moreover, BA stimulated ALA accumulation after illumination. This accumulation is more rapid than observed in the darkness in both controls and BA-treated cotyledons. In contrast to the results in the dark, the result when inhibitors of RNA and protein synthesis were present after illumination suggests that ALA accumulation and chlorophyll synthesis lead to an enhanced synthesis of m-RNA in the nucleus, and not activation of m-RNA with or without BA.

Formation of δ-aminolevulinic acid from glutamic acid by a partially purified enzyme system from wheat leaves

A method for partial purification of an enzyme system from greening wheat leaves which converts 14C-labeled glutamate to δ-aminolevulinic acid is described. The purification entails the successive use of anion and cation exchange, followed by moleculr sieving. The enzyme system is unstable in crude form, but the stability is markedly increased after column chromatography on CM-cellulose. The pH profile and the cofactor requirements suggest that at least two enzymes are involved.

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse, rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970) 208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F −, malonate, and arsenite), only F − (100 m m final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.

Induction of δ-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of delta-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.

New pathway for delta-aminolevulinic acid biosynthesis: formation from alpha-ketoglutaric acid by two partially purified plant enzymes.

Abstract Two enzymes which catalyze the formation of δ-aminolevulinic acid in two steps from α-ketoglutaric acid have been partially purified from Zea mays leaf extracts. The enzymes catalyze the following reactions: (1) a novel NADH-dependent reduction of the 1-carboxyl group of α-ketoglutarate, yielding 4,5-dioxovaleric acid, followed by (2) a transamination of this product with L-alanine to yield δ-aminolevulinate. The dehydrogenase cannot be demonstrated in crude extracts since it is masked by glutamic dehydrogenase. This pathway, in which the 5-carbon skeleton of α-ketoglutarate is utilized intact for δ-aminolevulinate formation, differs radically from the classical δ-aminolevulinate synthase reaction between glycine and succinyl-CoA.

Coordination of heme and globin synthesis in primate reticulocytes

In vitro indubations of the peripheral blood of marmosets, a new world monkey, have established the existence of an interaction between the heme and globin biosynthetic pathways in primate reticulocytes. In addition, isonicotinc acid hydrozide (INH), a known inhibitor of hem biosynthesis, was shown to inhibit secondarily the biosynthesis of globin. The effect of varied concentrations of INH on δ-[ 3H]-aminovelunic acid incorporation suggests that pyridoxal phosphate is required at two and possibly more steps in the heme pathway. The firts of such steps in at the condensation of succinyl-CoA and glycine; the second, most likely subsequent to the formation of δ-aminolevulinic acid, is at the mobilization of iron from the mitochondria. No significant accumulation of free globin was detected in the cells treated with INH.