Primer OKP71 (5′-ATAGCATGCAGCGGTTGAGTTAGGGCTTAAG-3′ [the SphI restriction site is underlined]) and mutagenic primer OKP73 (5′-GATTTGGTTTTTGATTTCAATGTTGGCAAATGTTCCTCTC-3′ [mutated residues are in boldface type]) were used to amplify the citB locus from 1,404 bp to 2,238 bp downstream of the start codon. F.M.M. Enhanced depolarization-evoked calcium signal and reduced [ATP]/[ADP] ratio are unrelated events induced by oxidative stress in synaptosomes. Values shown are the means and standard deviations from two technical replicates. This plasmid was introduced into E. coli by transformation, and the sequence was verified before introduction into B. subtilis strain AWS96 by a single crossover at the citB locus, producing strain KBP125 [citB+::pKP29(cat)]. The extent to which RNA processing also contributes to the diversity of citZ operon transcripts is not known. In the presence of inactivated aconitase, NAD(P)H fluorescence decreased only when α-KGDH was also inhibited (at higher H2O2 concentrations). (When KBP118 cultures were sampled and plated hourly between 2 and 5 h after inoculation, the proportion of suppressors/revertants increased from approximately 0.1% to 10% of the population over time.) Samples were taken at the indicated time points, and β-galactosidase activity was measured. Based on this structure, only one of the five residues mutated in the citB5 strain, Arg-741, has an IRP1 counterpart (Arg-728) in close proximity to the RNA. Primer pair FM167 (5′-GCGACACGCGGTCTTGAAGGG-3′) and FM168 (5′-CTAATACGACTCACTATAGGGAGAGGCGGATCAGACGGTTGTTGTC-3′ [the T7 promoter sequence is underlined]) was used to amplify the citZ open reading frame (ORF) from positions 7 to 1057. The hag RNA was used as a negative control. The role of oxidative abnormalities in the pathophysiology of Alzheimer's disease. Citrate is both cotransported with iron and a chelator of divalent cations, including iron. Although we hoped to isolate aconitase mutants that were selectively deficient in either enzymatic activity or RNA binding, both of the mutants that we have reported here are at least partially defective in both activities. This result provides an explanation for the result presented in Fig. The citZ gene is the first gene of an operon that includes the genes for isocitrate dehydrogenase (icd) and malate dehydrogenase (mdh). As described above, the enzyme activities of the two preparations were also different but inversely so (Fig. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. Expression was induced by the addition of 0.2% arabinose to exponentially growing, 500-ml cultures (OD600 = 0.6). Filters were washed twice with 0.5 ml reaction buffer, removed, and placed into scintillation fluid. 1B and Table 2). A radical hypothesis for neurodegeneration. The small RNA, whose synthesis is regulated by the motility sigma factor, σD, is suspected to act as an antisense RNA for citZ, but the effect of the RNA and its mechanism of action have not been elucidated. Analysis of the citZ 5′ UTR by using mfold (51) revealed several possible stem-loop structures that may form in vivo. Several arginine residues contribute to enzyme activity in IRP1 (55), but the homolog of Arg-741 in IRP1 (Arg-728) is not one of them. A “radical” view of cerebral ischemic injury. As expected, the citB null strain had very low levels of activity throughout growth (Fig. Thank you for sharing this Journal of Neuroscience article. A mock reaction mixture was added directly to scintillation fluid to obtain a measure of the total radioactivity of the RNA. Analysis of aconitase enzyme activity and expression levels in citB mutant strains. 8). Aconitase carries out the next stage of the cycle, an isomerisation from citrate to isocitrate, in which a hydroxy group moves from one carbon to the next. RNA retained on each membrane was detected by scintillation counting, and the fraction of RNA retained was calculated, after background subtraction, as a percentage of the input RNA. Aconitase (ACO) (genauer: Aconitat-Hydratase) heißen Enzyme, die die Umwandlung von Citrat oder Isocitrat in Aconitat und umgekehrt katalysieren. citB2 and citB7 mutants overexpress citB-lacZ and aconitase protein.To further explore this citrate accumulation phenotype, we monitored an expected side effect of citrate accumulation, i.e., increased expression from the citB promoter (9). While it is not as severe a defect as that of the C450S Acn protein, it is still surprising given the expectation that arginine-741 is not involved in enzyme activity. Increasing concentrations of purified Acn (0.25 to 4.5 μM) were incubated with a constant concentration of probe (250 nM) before analysis by polyacrylamide gel electrophoresis. Copyright © 2020 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 0021-9193; Online ISSN: 1098-5530, Two Roles for Aconitase in the Regulation of Tricarboxylic Acid Branch Gene Expression in, Sign In to Email Alerts with your Email Address. Extracts were analyzed by Western blotting using polyclonal antibodies raised to CS-II and CodY (loading control). Author information: (1)Department of Chemical Engineering, Columbia University , New York, New York 10027, United States. Gel mobility shift assays.To create RNA targets for gel mobility shift assays, the citZ leader region and hag transcript were amplified by PCR to generate templates for in vitro runoff transcription. Primer citBF6 (5′-GGGCATGCGAGAACCTCCTTAAAAGAGTTCGGTGTTATT-3′ [the SphI restriction site is underlined]) and mutagenic primer OKP37 (5′-GTTTGATGTATTTGTAGAGCTTGTAATCGCAGCAATGGC-3′ [mutated residues are in boldface type]) were used to amplify the citB locus from 400 bp upstream to 1,365 bp downstream of the start codon. Intracellular and extracellular citrate concentrations in citB mutant strainsb. Polyclonal antibodies raised in rabbits to B. subtilis aconitase (22), citrate synthase (34), isocitrate dehydrogenase (K. Matsuno and A. L. Sonenshein, unpublished data), and CodY (35) were used. Plants developed even more copies in mitochondria.Aconitase con… This is likely due to the lack of aconitase enzyme activity in these cells; the high citrate levels maintained in these mutants ensure that CcpC-dependent repression of citZ cannot be reinstated. Chloramphenicol-resistant transformants were isolated on DS medium and screened for spectinomycin sensitivity and glutamate auxotrophy. Only when α-KGDH is inhibited at higher concentrations of the oxidant (>50 μm) is the production of NADH compromised (Fig. The PCR product was digested with SacI and XbaI, and the resulting fragment was ligated to the expression vector pBAD30 (39); the resulting plasmid was named pFM1.