Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

delta-Aminolevulinic acid is the first committed precursor in the biosynthesis of hemes, phycobilins, and chlorophylls. Plants and algae synthesize delta-aminolevulinic acid from glutamate via an RNA-dependent 5-carbon pathway. Previous reports demonstrated that cyanobacteria form delta-aminolevulinic acid from glutamate in vivo. We now report the direct measurement of this activity in vitro. Three oxygenic prokaryotes were examined, the unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6) and the chlorophyll a- and b-containing filamentous prochlorophyte Prochlorothrix hollandica. delta-Aminolevulinic acid-forming activity was detected in soluble extracts of all three species. delta-Aminolevulinic acid formation by Synechocystis extracts was further characterized. Activity depended upon addition of reduced pyridine nucleotide, ATP, and Mg2+ to the incubation mixture. NADPH was a more effective pyridine nucleotide than NADH at low concentrations, but NADPH inhibited delta-amino-levulinic acid formation above 1 mM, whereas NADH did not. The pH optimum was about 7.6, and the ATP concentration optimum was 0.1 mM. Activity was stimulated by addition of RNA derived from Synechocystis or Chlorella, and abolished by preincubation with RNase A. After RNase inactivation, activity was restored by addition of RNasin to block further RNase action, followed by supplementation with Synechocystis RNA. Activity was inhibited by micromolar concentrations of hemin, as was previously found with plant and algal extracts. Complete dependence on added glutamate could not be achieved. Radioactivity was incorporated into delta-aminolevulinic acid when the incubation mixture contained 1-[14C]glutamate. Activity in the Synechocystis enzyme extract was stimulated by the addition of a partially purified enzyme fraction from Chlorella. It thus appears that prokaryotic oxygenic organisms share with chloroplasts the capacity for biosynthesis of photosynthetic pigments from glutamate via the RNA-dependent 5-carbon pathway.
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PMID:Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Synechocystis sp. PCC 6803 and other oxygenic prokaryotes. 245 30

Formation of the tetrapyrrole pigment precursor delta-aminolevulinic acid (ALA) from glutamate was detected and partially characterized in extracts of the strictly anaerobic green photosynthetic bacterial species Chlorobium vibrioforme by using assay methods derived from those developed for algae and cyanobacteria. ALA formation in Chlorobium extracts was saturated at 10 mM glutamate and required NADPH and ATP at optimal concentrations of 0.3 and 3 mM, respectively. Preincubation of the enzyme extract with RNase A destroyed the ALA-forming activity completely. Activity in the RNase-treated extract was restored by supplementation with Chlorobium RNA after addition of RNasin to block further RNase action. RNA from the cyanobacterium Synechocystis sp. strain PCC 6803 and Escherichia coli tRNAGlu also restored activity. Activity was inhibited 50% by 0.2 microM hemin. ALA formation was completely abolished by the addition of 5 microM 3-amino-2,3-dihydrobenzoic acid (gabaculine). These results indicate that Chlorobium extracts share with those of plants, eucaryotic algae, cyanobacteria, prochlorophytes, and methanogens the capacity for RNA-dependent ALA formation from glutamate.
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PMID:Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Chlorobium vibrioforme. 247 78

Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.
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PMID:delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA. 251 Oct 63

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA(Glu), ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the alpha subgroup of purple bacteria.
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PMID:Distribution of delta-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups. 278 25

Formation of delta-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 tRNAglu 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.
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PMID:RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris. 400 65

From Salmonella typhimurium LT2 hemA (delta-aminolevulinic acid requiring) 15 mutants were isolated which grew on the hydrophobic compound hemin. All had increased sensitivity to antibiotics such as vancomycin, bacitracin, novobiocin, erythromycin, rifampin, and oleandomycin, and were considered to be envelope mutants (Env-). Half the mutants were rough , based on altered bacteriophage sensitivity and deoxycholate sensitivity, whereas the remainder were smooth; three of the smooth mutants were studied in detail. They gave increased uptake of gentian violet but no increase in leakage of a periplasmic protein, RNase I. The total membranes and fractions from sucrose gradient centrifugations representing inner and outer membranes of the wild type and three mutants were examined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focussing - PAGE (IEF-PAGE). The major outer membrane proteins (molecular weights (MW)33 000, 34 000, 35 000, and 36 000) showed no or very little alterations in the Env- mutants. In SA1926 (env-52) one protein spot at MW 48 000, proven to be an outer membrane protein, was missing, whereas a new spot appeared nearby, and other proteins in this area of the gel were reduced. An Env+ transductant selected from this strain had the wild-type protein pattern restored. The two other Env- mutants had similar but not identical changes in protein composition.
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PMID:Isolation and characterization of hemin-permeable, envelope-defective mutants of Salmonella typhimurium. 701 21

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor delta-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3' portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.
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PMID:Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules. 995 55

Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).
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PMID:Evidence for direct interaction between enzyme I(Ntr) and aspartokinase to regulate bacterial oligopeptide transport. 1128 31

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.
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PMID:Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid. 1204 71

5-Aminolevulinic acid synthesis in isolated, intact, developing chloroplasts from greening cucumber (Cucumis sativus) cotyledons was inhibited by broken chloroplast fragments. It was shown that the inhibitory constituent was associated with the thylakoid membrane system. The inhibitor was resistant to boiling, was not a form of ribonuclease, and did not inhibit Mg-chelatase, indicating that massive organelle destruction was not involved. The inhibitor was also found in etioplast and mature chloroplasts; and it was found in barley as well as cucumber. 5-Aminolevulinate synthesis in the dark with exogenous ATP and NADPH, or in the light without added cofactors, were inhibited approximately equally. In the dark, 5-aminolevulinate synthesis and protochlorophyllide synthesis from glutamate were inhibited to about equal extent. The inhibition was decreased when the membranes were washed with aqueous acetone prior to incubation. The inhibition by the unknown factor was compared to the inhibition by gabaculine, 4-amino-5-hexynoic acid, protoheme, and glutathione. The unknown inhibitor appeared to have a number of similarities with protoheme.
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PMID:Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts : IV. An Endogenous Inhibitor from the Thylakoid Membranes. 1666 54


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