scholarly journals Protochlorophyllide reductase in photosynthetic prokaryotes and its role in chlorophyll synthesis

1995 ◽  
Vol 311 (2) ◽  
pp. 417-424 ◽  
Author(s):  
J D Rowe ◽  
W T Griffiths
Keyword(s):  
Dna Sequences ◽  
Specific Activity ◽  
Higher Plants ◽  
Red Light ◽  
Protein A ◽  
Chlorophyll Synthesis ◽  
Proteolytic Fragment ◽  
A Value ◽  
Protochlorophyllide Reductase ◽  
Reductase Gene

1. DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples. No such hybridization was observed with DNA from photosynthetic bacteria. 2. A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence. At the protein level the similarity is 91%. When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe. 3. Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations. These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively. 4. Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells. 5. The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants.

Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

2002 ◽  
Vol 362 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Johanna E. CORNAH ◽  
Jennifer M. ROPER ◽  
Davinder Pal SINGH ◽  
Alison G. SMITH
Keyword(s):  
Ferrous Iron ◽  
Specific Activity ◽  
Higher Plants ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Marker Enzyme ◽  
Hexane Extraction ◽  
Higher Plant ◽  
Major Site ◽  
Haem Biosynthesis

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

scholarly journals Characterization of the SARS-CoV-2 coronavirus X4-like accessory protein

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Olanrewaju Ayodeji Durojaye ◽  
Nkwachukwu Oziamara Okoro ◽  
Arome Solomon Odiba
Keyword(s):  
Rapid Development ◽  
Protein A ◽  
The Novel ◽  
Quality Model ◽  
A Value ◽  
Model Protein ◽  
Novel Coronavirus ◽  
Global Threat

Abstract Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

scholarly journals N-1-naphthylphthalamic acid stimulates tomato hypocotyl elongation, elevates ethylene levels and alters metabolic homeostasis

2018 ◽  
Author(s):  
Sapana Nongmaithem ◽  
Sameera Devulapalli ◽  
Yellamaraju Sreelakshmi ◽  
Rameshwar Sharma
Keyword(s):  
Butyric Acid ◽  
Metabolic Networks ◽  
Higher Plants ◽  
Red Light ◽  
Hypocotyl Elongation ◽  
Stimulated Emission ◽  
Ethylene Action ◽  
Naphthylphthalamic Acid ◽  
Exogenous Ethylene ◽  
Species Specific

One sentence summaryN-1-naphthylphthalamic acid (NPA) treatment stimulates tomato hypocotyl elongation likely by elevating ethylene emission and lowering indole-3-butyric acid levels in the seedlings.AbstractIn higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3-butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

scholarly journals Obstetric Outcome in Pregnant Patients with Low Level of Pregnancy-Associated Plasma Protein A in First Trimester

2018 ◽  
Vol 6 (6) ◽  
pp. 1028-1031 ◽  
Author(s):  
Vesna Livrinova ◽  
Igor Petrov ◽  
Igor Samardziski ◽  
Viktorija Jovanovska ◽  
Slagjana Simeonova-Krstevska ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Placental Abruption ◽  
Protein A ◽  
First Trimester ◽  
Target Group ◽  
Growth Restriction ◽  
Control Group ◽  
Unfavourable Outcome ◽  
Fetal Demise ◽  
A Value

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A), is a protease which releases Insulin-like growth factor. The role of this factor is stimulation of cell mitosis, differentiation and trophoblastic invasion of deciduas. Identification of patients with low PAPP-A (under 0.4 MoM in the first trimester has an influence on birth weight, attenuation of fetal growth, preeclampsia, birth and fetal demise.AIM: The main issue in the study is evaluating an influence of PAPP-A, calculated in the first trimester on the unfavourable outcome of pregnancy.MATERIAL AND METHODS: Seventy pregnant women with singleton pregnancy underwent first-trimester biochemical screening. The target group were women with PAPP-A below 0.4 MoM, and in control group, PAPP-A were оver 0.4 MoM. There was an assessment of the influence on the mode of delivery, gestational week, the presence of intrauterine growth restriction, preeclampsia, temporary birth, intrauterine fetal demise and newborn condition.RESULTS: In target group, consisted of 35 patients, 16 were delivered at term. From 28 to 37 g.w.- were 7 patient, 22-28 g.w.- 4 and 8 patients were under the 22 g.w (all with fetal demise) there were 19 pretemporary deliveries - 9 with Cesarean Section (SC). In the target group: 5 newborn wеre with IUGR, 6 women had preeclampsia, 1 had placental abruption. In control group were 35 patients: 28 delivered at term, 9 with SC, 26 vaginal deliveries; with IUGR were 4 newborns. Two newborns were hypertrophic.CONCLUSION: There is a significant difference in unfavourable outcome in the cases with PAPP-A under 0.4 Mom, particular in the group, with a PAPP-A value under 0.2 MoM. The patients delivered with SC with the main indications in utero hypoxia, growth restriction and elevated blood pressure had PAPP-A between 0.3-0.4 MoM. The patients with intrauterine fetal death and placental abruption in the most of the cases have PAPP-A value under 0.2 MoM. There is a need to be aware in these pregnancies to achieve the preventions of adverse outcome, to decrease perinatal morbidity and mortality.

Changes in protein composition of meiotic nodules during mammalian meiosis

1998 ◽  
Vol 111 (4) ◽  
pp. 413-423 ◽  
Author(s):  
A.W. Plug ◽  
A.H. Peters ◽  
K.S. Keegan ◽  
M.F. Hoekstra ◽  
P. de Boer ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Protein A ◽  
Reca Protein ◽  
Protein Composition ◽  
Homologous Chromosomes ◽  
E Coli ◽  
Recombination Nodules ◽  
Chromosome Synapsis ◽  
Repair Protein ◽  
Mismatch Repair Protein

Homologous chromosome synapsis and meiotic recombination are facilitated by several meiosis-specific structures: the synaptonemal complex (SC), and two types of meiotic nodules: (1) early meiotic nodules (MNs), also called zygotene nodules or early recombination nodules, and (2) late recombination nodules (RNs). The former are thought to be nucleoprotein complexes involved in the check for homology preceding, or accompanying synapsis, while the latter have been shown to be involved in reciprocal recombination. We have examined by immunocytochemistry the meiotic localization of a series of proteins at sites along the asynapsed axial elements prior to homologous synapsis and at sites along the SCs following synapsis. Several of the proteins examined have been implicated in repair/recombination and include RAD51, a mammalian homolog of the Escherichia coli RecA protein; Replication Protein-A (RPA), a single-strand DNA binding protein; and MLH1, a mismatch repair protein which is a homolog of the E. coli MutL protein. In addition two proteins were examined that have been implicated in meiotic checkpoints: ATM, the protein mutated in the human disease Ataxia Telangiectasia, and ATR, another member of the same family of PIK kinases. We present evidence that these proteins are all components of meiotic nodules and document changes in protein composition of these structures during zygonema and pachynema of meiotic prophase in mouse spermatocytes. These studies support the supposition that a subset of MNs are converted into RNs. However, our data also demonstrate changes in protein composition within the context of early MNs, suggesting a differentiation of these nodules during the process of synapsis. The same changes in protein composition occurred on both the normal X axis, which has no homologous pairing partner in spermatocytes, and on the axes of aberrant chromosomes that nonhomologously synapse during synaptic adjustment. These findings suggest that DNA sequences associated with MNs still must undergo an obligatory processing, even in the absence of interactions between homologous chromosomes.

Ubiquity and Diversity of Dissimilatory (Per)chlorate-Reducing Bacteria

1999 ◽  
Vol 65 (12) ◽  
pp. 5234-5241 ◽  
Author(s):  
John D. Coates ◽  
Urania Michaelidou ◽  
Royce A. Bruce ◽  
Susan M. O’Connor ◽  
Jill N. Crespi ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Contaminated Soils ◽  
Volatile Fatty Acids ◽  
Specific Activity ◽  
Paper Mill ◽  
Microbial Reduction ◽  
Cell Suspensions ◽  
Difference Spectra ◽  
Reducing Bacteria ◽  
Reductive Metabolism

ABSTRACT Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 × 103 to 2.4 × 106 cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 μmol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the generaPseudomonas and Azospirllum. However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.

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