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Query: UMLS:C1832526 (PCC)
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Membrane vesicles, isolated from osmotic lysates of Azotobacter vinelandii spheroplasts in Tris-acetate buffer, rapidly accumulate calcium in the presence of an oxidizable substrate. The addition of D-lactate to vesicles increases the rate of calcium uptake by 34-fold; L-malate, NADH, NADPH, and reduced phenazine methosulfate are nearly as effective as lactate. The intravesicular calcium pool which accumulates under these conditions is rapidly discharged by isotopic exchange or in the presence of respiratory inhibitors, uncouplers, or EGTA. The uptake rates for calcium follow Michaelis-Menten kinetics yielding a Km of 48 microM and a V max of 45 nmoles/min/mg membrane protein. Initial rates of EGTA-induced calcium efflux also follow saturation kinetics, giving a V max identical to that for calcium entry; but the Km for exodus is 14 mM, assuming that free calcium accumulates in vesicles. The difference in the affinity of calcium for the entry and exit processes observed during respiration is sufficient to account for the estimated 150-fold calcium concentration gradient achieved under steady-state conditions. The uptake system is specific for calcium as opposed to other cations, but zinc and lanthanum are effective competitors. Calcium uptake is blocked when electron is inhibited by exposure of vesicles to p-chlormercuriphenylsulfonate, hydroxyquinoline-N-oxide, or cyanide, or under anoxic conditions. Divalent cation ionophores (A23187 and X537A) and proton ionophores (CCP and gramicidin D) also block calcium transport effectively. The electrogenic potassium ionophore valinomycin has no effect on lactate-dependent calcium uptake in the presence of potassium; but ionophores which induce electroneutral exchange of protons for sodium or potassium (monensin and nigericin, respectively) did block calcium transport in the presence of the appropriate cation. The fluorescence intensity of quinacrine (an amine probe) in the presence of A. vinelandii membrane vesicles is reduced by 25% on addition of lactate; the quenching is blocked by CCP. This indicates that a pH gradient (inside acid) is developed across the vesicle membrane during lactate oxidation. These results indicate that these membrane preparations contain vesicles of inverted topology (with respect to the intact cell) and suggest that calcium transport occurs by means of electroneutral calcium/proton antiport.
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PMID:Respiration-coupled calcium transport by membrane vesicles from Azotobacter vinelandii. 11 11

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.
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PMID:Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. 173 Feb 47

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.
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PMID:Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase. 173 24

delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu). The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed. We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme. Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.
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PMID:Purification of glutamyl-tRNA reductase from Synechocystis sp. PCC 6803. 190 97

Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.
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PMID:Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis. 244 64

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

Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5'-flanking region and 430 base pairs of the 3'-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription.
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PMID:Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120. 755 23

The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.
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PMID:Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp. PCC 6803. Isolation and insertional inactivation of gltB and gltS genes. 772 52

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.
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PMID:ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803. 776 63


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