scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

scholarly journals Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

2015 ◽  
Vol 71 (4) ◽  
pp. 986-995 ◽  
Author(s):  
C. M. D. Swarbrick ◽  
M. A. Perugini ◽  
N. Cowieson ◽  
J. K. Forwood
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Size Exclusion ◽  
X Ray Scattering ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Enzyme Families

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on theEscherichia colistructure.Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of theY. pestisTesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

scholarly journals Isolation and biochemical characterization of the α- and β-subunits of glycoprotein IIb of human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Size Exclusion ◽  
Beta Subunits ◽  
Exclusion Chromatography ◽  
Stepwise Reduction ◽  
Acidic Nature

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

scholarly journals Impact of crystal packing on coiled-coil flexibility.

2014 ◽  
Vol 70 (a1) ◽  
pp. C1599-C1599
Author(s):  
François Ferron ◽  
David Blocquel ◽  
Johnny Habchi ◽  
Eric Durand ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Measles Virus ◽  
Quaternary Structure ◽  
Crystal Packing ◽  
Coiled Coil ◽  
Size Exclusion ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Partial Unfolding

The structural characterization of various constructs of the Measles virus (MeV) Phosphoprotein (P) multimerization domain (PMD) has brought to light significant discrepancies in the quaternary structure due to both crystal constraints and the flexible nature of this coiled-coil. Indeed, despite a conserved tetrameric parallel coiled-coil core, structural comparison unveiled significant deformations in the C-terminal extremities that even led to the partial unfolding of the coiled-coil. These deformations were induced by intermolecular interactions within the crystal, as well as by the crystallization condition. These deformations also suggest that PMD has the ability to adapt to external mechanical constrains. Using a combination of biophysical methods (size-exclusion chromatography, circular dichroism and small angle X-ray scattering), we assessed the differential flexibility of the C-terminal region of the MeV PMD in solution. Taken together, these results show that crystal packing can be used to "freeze" in a certain state, parts of proteins known to be in a dynamic folding-unfolding equilibrium. They also bring awareness that conclusions about function and mechanism based on analysis of a single crystal structure of a known dynamic protein can be easily biased, and they challenge to some extent the assumption that coiled-coil structures can be reliably predicted from the amino acid sequence.

scholarly journals Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

2005 ◽  
Vol 48 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Maria Aparecida Souza ◽  
Francielle Amâncio-Pereira ◽  
Cristina Ribeiro Barros Cardoso ◽  
Adriano Gomes da Silva ◽  
Edmar Gomes Silva ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Terminal Sequence ◽  
Native Page ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Binding Lectin

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

scholarly journals Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 582
Author(s):  
Cristian Franco-Servín ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
Ramsés Alejandro Rosales-García ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Biochemical Characterization ◽  
Two Dimensions ◽  
Size Exclusion ◽  
Muscle Twitch ◽  
Amino Terminal ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Main Components

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

2021 ◽  
Author(s):  
Quynh DangThu ◽  
Thu-Thuy Nguyen ◽  
Sei-Heon Jang ◽  
ChangWoo Lee
Keyword(s):  
Biochemical Characterization ◽  
Size Exclusion ◽  
Sorbitol Dehydrogenase ◽  
High Catalytic Activity ◽  
Active Site Residues ◽  
Tertiary Structures ◽  
Cold Adapted ◽  
Pseudomonas Mandelii ◽  
Catalytic Active Site ◽  
Exclusion Chromatography

Abstract Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents, and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized D-sorbitol as its carbon source, among the four polyols examined (D-galactitol, D-mannitol, D-sorbitol, or D-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153, and Lys157 as catalytic active site residues and existed as a ∼67 kDa dimer in size-exclusion chromatography. PmSDH converted D-sorbitol to D-fructose using NAD+ as a coenzyme and, vice versa, D-fructose to D-sorbitol using NADH as a coenzyme. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. At 40°C, PmSDH was rapidly denatured. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well-suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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