scholarly journals The Cryptic Plastid of Euglena longa Defines a New Type of Nonphotosynthetic Plastid Organelle

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Zoltán Füssy ◽  
Kristína Záhonová ◽  
Aleš Tomčala ◽  
Juraj Krajčovič ◽  
Vyacheslav Yurchenko ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
Physiological Role ◽  
Redox Balance ◽  
Redox Status ◽  
Secondary Endosymbiosis ◽  
Content Type ◽  
Metabolic Functions ◽  
Green Algal ◽  
Thioredoxin System ◽  
Algal Groups

ABSTRACT Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa, we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast. IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis. We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa. Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

scholarly journals The cryptic plastid of Euglena longa defines a new type of non-photosynthetic plastid organelles

2019 ◽  
Author(s):  
Zoltán Füssy ◽  
Kristína Záhonová ◽  
Aleš Tomčala ◽  
Juraj Krajčovič ◽  
Vyacheslav Yurchenko ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
Physiological Role ◽  
Redox Status ◽  
Secondary Endosymbiosis ◽  
Metabolic Functions ◽  
Green Algal ◽  
Photosynthetic Eukaryotes ◽  
Algal Groups ◽  
New Type ◽  
Green Flagellate

AbstractMost secondarily non-photosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close non-photosynthetic relative of Euglena gracilis harbouring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in biosynthesis of isoprenoid precursors and fatty acids, and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalysed by the SqdX form of the enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for non-photosynthetic plastids studied so far. The most striking attribute of the organelle is the presence of a linearized Calvin-Benson (CB) pathway including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin systems. We hypothesize that FNR passes electrons to the ferredoxin/thioredoxin systems from NADPH to activate the linear CB pathway in response to the redox status of the E. longa cell. In effect, the pathway may function as a redox valve bypassing the glycolytic oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best studied organelle of this category, the apicoplast.ImportanceColourless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here we study the elusive plastid of Euglena longa, a non-photosynthetic cousin of the familiar green flagellate Euglena gracilis. We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized non-photosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa. Our work provides a detailed account on a unique variant of relic plastids, the first among non-photosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to non-photosynthetic plastids.

scholarly journals Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

2014 ◽  
Vol 197 (2) ◽  
pp. 296-306 ◽  
Author(s):  
Constanze Pinske ◽  
Monique Jaroschinsky ◽  
Sabine Linek ◽  
Ciarán L. Kelly ◽  
Frank Sargent ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Hydrogen Oxidation ◽  
Physiological Role ◽  
Redox Status ◽  
Content Type ◽  
Whole Cells ◽  
Fumarate Reduction ◽  
Hydrogenase 2 ◽  
Quinone Pool

Escherichia coliuptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochromebmembrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2oxidation or H2evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2evolution from glycerol requires reverse electron transport.

scholarly journals Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Maude F. Lévêque ◽  
Laurence Berry ◽  
Michael J. Cipriano ◽  
Hoa-Mai Nguyen ◽  
Boris Striepen ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Growth Conditions ◽  
Model Organisms ◽  
Secondary Endosymbiosis ◽  
Related Protein ◽  
Content Type ◽  
Superresolution Microscopy ◽  
Animal Pathogens ◽  
Cellular Components ◽  
Catabolic Process

ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

scholarly journals Why Orange Guaymas Basin Beggiatoa spp. Are Orange: Single-Filament-Genome-Enabled Identification of an Abundant Octaheme Cytochrome with Hydroxylamine Oxidase, Hydrazine Oxidase, and Nitrite Reductase Activities

2012 ◽  
Vol 79 (4) ◽  
pp. 1183-1190 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Jennifer F. Biddle ◽  
Jason R. Siebert ◽  
Eric Staunton ◽  
Eric L. Hegg ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Pure Culture ◽  
Gulf Of California ◽  
Microbial Mats ◽  
Physiological Role ◽  
Nitrite Reduction ◽  
Cold Seeps ◽  
Single Filament ◽  
Guaymas Basin ◽  
Content Type

ABSTRACTOrange, white, and yellow vacuolatedBeggiatoaceaefilaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolateBeggiatoaceaeare yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orangeBeggiatoa(“CandidatusMaribeggiatoa”) filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (μLC–MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown byin vitroassays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known ofBeggiatoaceaephysiology, nitrite reduction is the most likelyin vivorole of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

scholarly journals Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica

2010 ◽  
Vol 9 (6) ◽  
pp. 926-933 ◽  
Author(s):  
Mohammad Abu Yousuf ◽  
Fumika Mi-ichi ◽  
Kumiko Nakada-Tsukui ◽  
Tomoyoshi Nozaki
Keyword(s):  
Entamoeba Histolytica ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Protozoan Parasite ◽  
Pyridine Nucleotide ◽  
Immunofluorescence Assay ◽  
Chemical Degradation ◽  
Content Type ◽  
Targeting Signal ◽  
Pyridine Nucleotide Transhydrogenase

ABSTRACT Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.

Single nucleotide polymorphism rs1128446 ТXNRD1 is a novel genetic marker of cerebral stroke

2021 ◽  
pp. 37-43
Author(s):  
О.Ю. Бушуева ◽  
А.В. Полоников ◽  
В.П. Иванов
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Ischemia Reperfusion ◽  
Basic Mechanism ◽  
Bioinformatic Analysis ◽  
Redox Status ◽  
Cerebral Stroke ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Increased Risk ◽  
Thioredoxin System

Мозговой инсульт (МИ) занимает третье место в структуре смертности во всем мире и является ведущим фактором снижения когнитивных функций и деменции. Окислительный стресс является ведущим механизмом повреждения головного мозга при ишемии и последующей реперфузии. Тиоредоксиновая система является наиболее важным антиоксидантным барьером клетки, способным регулировать ее окислительно-восстановительный статус. Целью исследования было изучение ассоциации однонуклеотидного полиморфизма rs1128446 гена эндогенного регулятора тиоредоксина ТXNRD1 с риском развития МИ. Материалом для исследования послужила выборка неродственных жителей Центральной России общей численностью 825 человек. В исследование были включены 375 пациентов с МИ (216 мужчин, 159 женщин; средний возраст 59,44±0,51 лет). Контрольную группу составили 450 относительно здоровых индивидуумов (249 мужчин, 201 женщина, средний возраст 61,69±0,38 лет) без кардио- и цереброваскулярных заболеваний в анамнезе и имеющих нормальный уровень артериального давления. Генотипирование SNP проводили методом ПЦР в режиме реального времени путем дискриминации аллелей с помощью TaqMan-зондов. Для анализа ассоциаций генотипов с развитием заболевания пользовались лог-аддитивной регрессионной моделью. Все расчеты выполнены относительно минорного аллеля; введены поправки на пол и возраст. SNP rs1128446 ТXNRD1 был связан с повышенным риском развития МИ (OR=1,89; 95% CI=1,48-2,43; p<0,0001). Проведенный биоинформатический анализ выявил высокий регуляторный потенциал данного SNP в тканях сердечно-сосудистой системы. Таким образом, впервые установлена ассоциация rs1128446 ТXNRD1 с развитием МИ. Cerebral stroke (CS) is the leading factor in cognitive decline and dementia and ranks third in the structure of mortality worldwide. Oxidative stress is the basic mechanism of brain damage after cerebral ischemia-reperfusion. The thioredoxin system is the most important antioxidant barrier of the cell, capable of regulating its redox status. The aim of this study was to investigate the association of the common single nucleotide polymorphism rs1128446 in gene encoding the endogenous thioredoxin regulator TXNRD1 with the risk of CS. A total of 825 unrelated individuals from Central Russia were included for this study: 375 patients with CS (216 males, 159 females; 59.44±0.51 years old) and 450 healthy controls (249 males, 201 females, 61.69±0.38 years old). Genotyping was performed using TaqMan-based PCR. To analyze the associations of genotypes with the risk of diseases, a log-additive regression model was used. All calculations were performed relative to the minor allele; corrections for gender and age have been introduced. SNP rs1128446 TXNRD1 was associated with an increased risk of CS (OR=1.89; 95% CI=1.48-2.43; P<0.0001). Bioinformatic analysis revealed a high regulatory potential of this SNP in tissues of the cardiovascular system. Thus, for the first time, the association of rs1128446 TXNRD1 with the development of CS was revealed.

scholarly journals Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors

Antioxidants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 129
Author(s):  
Yuta Hatori ◽  
Takanori Kubo ◽  
Yuichiro Sato ◽  
Sachiye Inouye ◽  
Reiko Akagi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Biological Membrane ◽  
Redox Homeostasis ◽  
Redox Balance ◽  
Redox Status ◽  
Golgi Body ◽  
Redox Equilibrium ◽  
Cytosolic Glutathione ◽  
Redox Sensors ◽  
Subcellular Resolution

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

scholarly journals Draft Genome Sequence of the Industrially Significant Bacterium Pseudomonas fluorescens ATCC 13525

2018 ◽  
Vol 7 (17) ◽  
Author(s):  
M. J. Meier ◽  
R. M. Subasinghe ◽  
L. A. Beaudette
Keyword(s):  
Antibiotic Resistance ◽  
Pseudomonas Fluorescens ◽  
Virulence Factors ◽  
Draft Genome ◽  
Antibiotic Resistance Genes ◽  
Strain Atcc ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Content Type ◽  
Metabolic Functions

Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolic functions and potential industrial uses. We sequenced P. fluorescens strain ATCC 13525 with the goal of determining virulence factors and antibiotic resistance genes to predict the potential impacts on human and environmental health in the event of exposure.

scholarly journals Acid Stress-Mediated Metabolic Shift in Lactobacillus sanfranciscensis LSCE1

2011 ◽  
Vol 77 (8) ◽  
pp. 2656-2666 ◽  
Author(s):  
Diana I. Serrazanetti ◽  
Maurice Ndagijimana ◽  
Sylvain L. Sado-Kamdem ◽  
Aldo Corsetti ◽  
Rudi F. Vogel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Acid Stress ◽  
Redox Balance ◽  
Gas Chromatography Mass Spectrometry ◽  
Metabolic Shift ◽  
Content Type ◽  
Lactobacillus Sanfranciscensis ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTLactobacillus sanfranciscensisLSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth inL. sanfranciscensisin restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.

scholarly journals Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis

1987 ◽  
Vol 247 (3) ◽  
pp. 679-685 ◽  
Author(s):  
F Watanabe ◽  
Y Nakano ◽  
S Kitaoka
Keyword(s):  
Euglena Gracilis ◽  
Binding Proteins ◽  
Microsomal Fraction ◽  
Binding Protein ◽  
Physiological Role ◽  
Binding Activity ◽  
Thiol Groups ◽  
Double Immunodiffusion ◽  
Ph Dependency ◽  
Ks Values

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

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