Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells

2017 ◽  
Vol 474 (10) ◽  
pp. 1689-1703 ◽  
Author(s):  
Frédéric Toussaint ◽  
Baptiste Pierman ◽  
Aurélie Bertin ◽  
Daniel Lévy ◽  
Marc Boutry
Keyword(s):  
Electron Microscopy ◽  
Drug Resistance ◽  
Nicotiana Tabacum ◽  
Abc Transporters ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Pleiotropic Drug Resistance ◽  
Suspension Cells

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia, which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min−1 mg−1) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters.

Plant Pathology Diagnosed by X-Ray Microanalysis

1987 ◽  
Vol 45 ◽  
pp. 974-975
Author(s):  
J. H. Resau ◽  
N. Howell ◽  
S. H. Chang
Keyword(s):  
Electron Microscopy ◽  
Plant Pathology ◽  
Biochemical Characterization ◽  
Nutrient Deficiency ◽  
Green Leaf ◽  
Parasitic Infestation ◽  
X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Genome-Wide Screening and Genetic Networks in Pleiotropic Drug Resistance, Oxidative Stress, and Drug Targets in S. Cerevisiae

2021 ◽  
Author(s):  
◽  
Ploi Yibmantasiri
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Abc Transporters ◽  
Oxidative Stress Response ◽  
Genetic Networks ◽  
Antifungal Compound ◽  
Pleiotropic Drug Resistance ◽  
Gene Deletions ◽  
Genome Wide ◽  
And Oxidative Stress

<p>One of the major problems in biology is to identify genes that are involved in specific processes. Classical genetics and biochemistry, although powerful and informative, can be very labour intensive and do not necessarily characterise networked genes in processes that may overarch numerous biochemical pathways. Here we utilised genomic tools that are capable of defining networks to identify genes involved the complex target mode-of-action of a novel antifungal compound, neothyonidioside and in regulating specific stress processes and the PDR phenotype. The first part of this study investigated the mode-of-action of the antifungal compound, neothyonidioside (neo). We developed a neo resistant mutant strain then utilising a modification of SGAM, a genetic mapping tool, and application of genome-wide chemical-genetic profiling, we identified the neo resistant locus NCP1. This gene acts at a late step in ergosterol biosynthesis but is not the target of neo. The finding that many of the component genes in the ESCRT complex were necessary for neo resistance allowed us to predict and verify by high-content fluorescence microcopy that interruptions in the endosome-multivesicular body pathway were involved. From the known function of the ESCRT proteins and that neo binds ergosterol only above threshold concentrations of ergosterol (explaining the mutant phenotype) we concluded that neo disruption of membrane curvature and fusion capability in the endosome-vacuole pathway is its target. In the second part of this study we identified genes in a genome-wide fashion that modulate the pleiotropic drug resistance (PDR) phenotype and oxidative stress response. Many PDR targets are well studied ABC transporters (e.g. PDR5 , YOR1), but the modulating events between xenobiotic sensing and transcription factor activation, and possible crosstalk between PDR and other stress responses such as oxidative stress are not well characterised. To identify specific genes involved in the PDR and oxidative stress processes, we developed a fluorescent reporter screen for effects on the PDR-target ABC-transporters, Pdr5p and Yor1p tagged with GFP. For the oxidative stress response, the oxidative stress (OS) transcription factor Yap1p tagged with GFP was used. Each reporter was placed in the yeast non-essential gene deletion background of ~4800 strains which were then subjected to either xenobiotic treatments (PDR –GFP reporters) or oxidant treatments (Yap1p-GFP). We then screened for gene deletions which prevented the normal upregulation of PDR reporters in the presence of xenobiotics. Controls were included in the screens that assured we were assessing genes that must contribute to or act before the transcription of the ABC-transporters. A similar screening strategy was pursued for identifying gene deletions that prevent the normal nuclear re-localisation of Yap1p in the presence of oxidants. A major finding in this study was identification of genes contributing to the PDR phenotype that involved signalling (Rho-GTPase, MAPK), that were involved in RNA polymerase II mediator complexes and chromatin modification (subunits of ADA and SAGA histone acetyltransferase complexes), and that were involved in sphingo/phosphorlipids biosynthesis. Secondary screens comprising spot dilution growth assays and Western blots of Pdr5p abundance confirmed key genes of the primary screen and showed that these were specific and not global transcriptional effects.For some of the gene-dependencies, our results can only be construed to indicate the existence of alternative pathways underpinning the PDR phenotype in a Pdr1p/Pdr3p independent manner. We then supposed that if in fact PDR phenotypes are the result of genetic networks, then genes known to interact with the most highly connected hubs from our PDR screen results should also to some extent contribute to the PDR phenotype (spot dilution growth assays, Western blot abundance). A selection of 18 such genes that also appeared in our primary screen but were deemed to be below the cut-off point were phenotype tested and in 60% of the cases showed similar phenotypes to the genes already identified. This result not only proved the validity of the screening methods but validated the original supposition, i.e. that PDR phenotypes can be affected, through gene networks.</p>

scholarly journals New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

2002 ◽  
Vol 22 (8) ◽  
pp. 2642-2649 ◽  
Author(s):  
Stéphane Le Crom ◽  
Frédéric Devaux ◽  
Philippe Marc ◽  
Xiaoting Zhang ◽  
W. Scott Moye-Rowley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Binding Site ◽  
Time Course ◽  
Target Genes ◽  
Regulation System ◽  
Dependent Manner ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

2018 ◽  
Vol 43 (6) ◽  
pp. 638-650
Author(s):  
Ruth Ololade Amiola ◽  
Adedeji Nelson Ademakinwa ◽  
Zainab Adenike Ayinla ◽  
Esther Nkechi Ezima ◽  
Femi Kayode Agboola
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Subunit Molecular Weight ◽  
Cyanoalanine Synthase ◽  
Germinating Seeds ◽  
Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria

2020 ◽  
Author(s):  
Juliane Zimmerling ◽  
Michel Oelschlägel ◽  
Carolin Großmann ◽  
Matthias Voitel ◽  
Michael Schlömann ◽  
...  
Keyword(s):  
Esterase Activity ◽  
Soil Bacteria ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Aldehyde Dehydrogenases ◽  
Long Term Stability ◽  
Almost All ◽  
Different Characteristics

Abstract Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

scholarly journals Purification and Biochemical Characterization of Alkaline Serine Protease from Caesalpinia bonducella

2010 ◽  
Vol 5 (6) ◽  
pp. 1934578X1000500
Author(s):  
Hidayatullah Khan ◽  
Irshad Ali ◽  
Arif-ullah Khan ◽  
Mushtaq Ahmed ◽  
Zamarud Shah ◽  
...  
Keyword(s):  
Serine Protease ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Alkaline Serine Protease ◽  
Protease Enzyme ◽  
Caesalpinia Bonducella

A high molecular weight serine protease has been purified to electrophoretic homogeneity from the seeds of Caesalpinia bonducella Flem. (Caesalpiniaceae) by the combination of size exclusion and ion exchange chromatography. About 524 fold purification was achieved with an overall recovery of 6.8%. The specific activity was found to be 86 U/mg/min at pH 8.0. The calculated Km and Vmax were 1.66 mg/mL and 496.68 units/min per mg of protein, respectively. The molecular mass was estimated to be about 63 kDa by sodium dodecyl sulfate PAGE. The enzyme showed optimum activity at pH 8.0 and exhibited its highest activity at 40°C. The enzyme was strongly inhibited by 2mM phenylmethylsulfonyl fluoride (PMSF), suggesting the presence of a serine residue at the active site. PMSF showed a pure competitive type of inhibition with the serine protease enzyme. It was observed that enzyme activity was enhanced in the presence of dications and was active against a variety of modified substrates and natural proteins.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes

1988 ◽  
Vol 255 (5) ◽  
pp. G579-G586
Author(s):  
B. Schjoldager ◽  
S. P. Powers ◽  
L. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Peptide Hormone ◽  
Molecular Probes ◽  
Affinity Labeling ◽  
Molecular Heterogeneity ◽  
Cck Receptors ◽  
Amino Terminal ◽  
Binding Subunit

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.

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