Molecular cloning and biochemical characterization of rabbit factor XI

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.

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

Toxins ◽

10.3390/toxins13080582 ◽

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.

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Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000486757 ◽

2018 ◽

Vol 28 (1) ◽

pp. 14-27 ◽

Cited By ~ 1

Author(s):

Carlos Eduardo Serrano-Maldonado ◽

Israel García-Cano ◽

Augusto González-Canto ◽

Eliel Ruiz-May ◽

Jose Miguel Elizalde-Contreras ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Food Industry ◽

Pathogenic Bacteria ◽

Heterologous Protein ◽

Biochemical Characterization ◽

Ph Values ◽

Food Borne ◽

Sds Page ◽

Exclusion Chromatography

The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62–75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6–7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

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Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Microbiology ◽

10.1099/mic.0.26414-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2455-2462 ◽

Cited By ~ 95

Author(s):

Masaru Nagai ◽

Maki Kawata ◽

Hisayuki Watanabe ◽

Machiko Ogawa ◽

Kumiko Saito ◽

...

Keyword(s):

Lentinula Edodes ◽

Veratryl Alcohol ◽

Size Exclusion ◽

Melanin Synthesis ◽

Sds Page ◽

Diammonium Salt ◽

Exclusion Chromatography ◽

Homogeneous Preparation

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

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Purification and partial biochemical characterization of normal human interleukin 1.

Journal of Experimental Medicine ◽

10.1084/jem.160.3.772 ◽

1984 ◽

Vol 160 (3) ◽

pp. 772-787 ◽

Cited By ~ 39

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.

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Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan

The Open Food Science Journal ◽

10.2174/1874256401810010033 ◽

2018 ◽

Vol 10 (1) ◽

pp. 33-45

Author(s):

Syed Abid Ali ◽

Fozia Humayun ◽

Iqra Munir ◽

Shakil Ahmad ◽

Zarrien Ayub ◽

...

Keyword(s):

Total Protein ◽

Biochemical Characterization ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Nutritional Composition ◽

The Body ◽

Size Exclusion ◽

High Concentration ◽

Sds Page

Objective: The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three major Thais species (T. bufo, T. hippocastanum and T. rudolphi) from the Sindh Coast, Pakistan. Methods: Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA & Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis. Results: Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the three Thais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmed via SDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined. Conclusion: Our findings suggest that Thais sp. are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

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

10.26434/chemrxiv.13110602 ◽

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

Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in Escherichia coli 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.

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Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100680118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2100680118

Author(s):

Anastasia C. Manesis ◽

Richard J. Jodts ◽

Brian M. Hoffman ◽

Amy C. Rosenzweig

Keyword(s):

Biochemical Characterization ◽

Protein A ◽

Copper Ion ◽

Copper Homeostasis ◽

Metabolic Enzyme ◽

Copper Binding ◽

Methane Oxidizing Bacteria ◽

And Function

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

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

Biochemical Journal ◽

10.1042/bj2400155 ◽

1986 ◽

Vol 240 (1) ◽

pp. 155-161 ◽

Cited By ~ 14

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.

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Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana)

Biochemical Journal ◽

10.1042/bj2720721 ◽

1990 ◽

Vol 272 (3) ◽

pp. 721-726 ◽

Cited By ~ 52

Author(s):

V L Koshte ◽

W van Dijk ◽

M E van der Stelt ◽

R C Aalberse

Keyword(s):

Cell Proliferation ◽

Affinity Chromatography ◽

Molecular Mass ◽

Size Exclusion ◽

T Cell Proliferation ◽

Isolation And Characterization ◽

Sds Page ◽

Exclusion Chromatography ◽

Binding Lectin

A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.

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