Characterization of sarcoplasmic protein in the meat using SDS-PAGE method

Background: Deinagkistrodon acutus (D. acutus) and Bungarus multicinctus (B. multicinctus) as traditional medicines have been used for hundreds of years in China. The venoms of these two species have strong toxicity on the victims. Objective: The objective of this study is to reveal the profile of venom proteins and peptides of D. acutus and B. multicinctus. Method: Ultrafiltration, SDS-PAGE coupled with in-gel tryptic digestion and Liquid Chromatography- Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) were used to characterize proteins and peptides of venoms of D. acutus and B. multicinctus. Results: In the D. acutus venom, 67 proteins (16 protein families) were identified, and snake venom metalloproteinases (SVMPs, 38.0%) and snake venom C-type lectins (snaclecs, 36.7%) were dominated proteins. In the B. multicinctus venom, 47 proteins (15 protein families) were identified, and three-finger toxins (3FTxs, 36.3%) and Kunitz-type Serine Protease Inhibitors (KSPIs, 32.8%) were major components. In addition, both venoms contained small amounts of other proteins, such as Snake Venom Serine Proteinases (SVSPs), Phospholipases A2 (PLA2s), Cysteine-Rich Secreted Proteins (CRISPs), 5'nucleotidases (5'NUCs), Phospholipases B (PLBs), Phosphodiesterases (PDEs), Phospholipase A2 Inhibitors (PLIs), Dipeptidyl Peptidases IV (DPP IVs), L-amino Acid Oxidases (LAAOs) and Angiotensin-Converting Enzymes (ACEs). Each venom also had its unique proteins, Nerve Growth Factors (NGFs) and Hyaluronidases (HYs) in D. acutus, and Cobra Venom Factors (CVFs) in B. multicinctus. In the peptidomics, 1543 and 250 peptides were identified in the venoms of D. acutus and B. multicinctus, respectively. Some peptides showed high similarity with neuropeptides, ACE inhibitory peptides, Bradykinin- Potentiating Peptides (BPPs), LAAOs and movement related peptides. Conclusion: Characterization of venom proteins and peptides of D. acutus and B. multicinctus will be helpful for the treatment of envenomation and drug discovery.

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Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19980434 ◽

1998 ◽

Vol 63 (3) ◽

pp. 434-440 ◽

Cited By ~ 2

Author(s):

Irena Hulová ◽

Jana Barthová ◽

Helena Ryšlavá ◽

Václav Kašička

Keyword(s):

Concanavalin A ◽

Reversed Phase ◽

Amino Acid Sequences ◽

Gel Chromatography ◽

Affinity Ligand ◽

Sds Page ◽

Terminal Amino ◽

Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

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Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00125-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Preeti Anand ◽

Jay Prakash Pandey ◽

Dev Mani Pandey

Keyword(s):

San Francisco ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Evolutionary Genetics ◽

Imaging Analysis ◽

Silk Cocoon ◽

Natural Color ◽

Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

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Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

Biochemical Journal ◽

10.1042/bj20041053 ◽

2005 ◽

Vol 387 (1) ◽

pp. 271-280 ◽

Cited By ~ 58

Author(s):

Seonghun KIM ◽

Sun Bok LEE

Keyword(s):

Molecular Mass ◽

Gel Filtration ◽

Maximal Activity ◽

Genome Database ◽

Sds Page ◽

Key Enzyme ◽

Bivalent Metal Ions ◽

Ammonium Sulphate Fractionation ◽

Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi

Canadian Journal of Microbiology ◽

10.1139/w06-019 ◽

2006 ◽

Vol 52 (7) ◽

pp. 651-657 ◽

Cited By ~ 49

Author(s):

Luis Morales de la Vega ◽

J Eleazar Barboza-Corona ◽

Maria G Aguilar-Uscanga ◽

Mario Ramírez-Lepe

Keyword(s):

Bacillus Thuringiensis ◽

Sodium Dodecyl ◽

Sclerotium Rolfsii ◽

Phytopathogenic Fungi ◽

Purification And Characterization ◽

Chitinolytic Enzyme ◽

Bacillus Thuringiensis Subsp ◽

Sds Page ◽

Fusarium Sp

A chitinolytic enzyme from Bacillus thuringiensis subsp. aizawai has been purified and its molecular mass was estimated ca. 66 kDa by sodium dodecyl sulfate – polyacryamide gel electrophoresis (SDS–PAGE). The enzyme was able to hydrolyze chitin to chitobiosides but not carboxymethylcellulose, cellulose, pullulan, and laminarin. Optimal pH and temperature were detected at 6 and 50 °C, respectively. Stability, in the absence of substrate, was observed at temperatures less than 60 °C and pH between 5 and 8. Enzyme activity was significantly inhibited by K+ and EDTA and completely inhibited by Hg2+. Purified chitinase showed lytic activity against cell walls from six phytopathogenic fungi and inhibited the mycelial growth of both Fusarium sp. and Sclerotium rolfsii. The biocontrol efficacy of the enzyme was tested in the protection of bean seeds infested with six phytopathogenic fungi.Key words: chitinase, Bacillus thuringiensis, purification, phytopathogenic fungi.

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Data on the characterization of native soy globulin by SDS-Page, light scattering and titration

Data in Brief ◽

10.1016/j.dib.2016.10.016 ◽

2016 ◽

Vol 9 ◽

pp. 749-752 ◽

Cited By ~ 5

Author(s):

Nannan Chen ◽

Mouming Zhao ◽

Christophe Chassenieux ◽

Taco Nicolai

Keyword(s):

Light Scattering ◽

Sds Page

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Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules

Journal of Cell Science ◽

10.1242/jcs.88.4.453 ◽

1987 ◽

Vol 88 (4) ◽

pp. 453-466

Author(s):

R.W. Linck ◽

M.J. Goggin ◽

J.M. Norrander ◽

W. Steffen

Keyword(s):

Immunofluorescence Microscopy ◽

Strongylocentrotus Purpuratus ◽

Body Region ◽

Two Dimensional ◽

Structural Localization ◽

Lytechinus Pictus ◽

Sds Page ◽

Nuclear Structures ◽

Sea Urchin Sperm

Rabbit antibodies raised and purified against three tektins, proteins of flagellar doublet microtubules from sea-urchin sperm (Lytechinus pictus and Strongylocentrotus purpuratus), were used to study tektin biochemistry and their structural localization. Doublet microtubules were fractionated into tektin filaments and separated by SDS-PAGE into three major tektin polypeptide bands (Mr = 47, 51 and 55 (X 10(3)), which were used to immunize rabbits. Antibodies against each tektin (anti-tektins) were affinity-purified and then characterized by two-dimensional isoelectric focusing/SDS-PAGE immunoblotting and by immunofluorescence microscopy. In two-dimensional immunoblots of 0.5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55 X 10(3) Mr tektin (anti-55) stained one major polypeptide of 55 X 10(3) Mr and pI 6.9, anti-51 stained two polypeptides of 51 X 10(3) Mr and pI approximately 6.15, and anti-47 stained one major polypeptide of 47 X 10(3) Mr and pI 6.15. The anti-tektins also stained several minor neighbouring polypeptides, which may be isoelectric variants, novel tektins or unrelated proteins. Furthermore, anti-47 crossreacted with the major 55 X 10(3) Mr polypeptide. By immunofluorescence microscopy all three anti-tektins stained methanol-fixed echinoderm sperm flagella and embryonic cilia. In addition, anti-47 and anti-55 stained unfixed, demembranated axonemes. Besides staining axonemes, all anti-tektins labelled the basal body region, and anti-51 labelled the sperm head envelope. These results indicate that the tektins are a complex family of proteins that are components of axonemal microtubules and possibly other cytoplasmic and nuclear structures.

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Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86821996000500012 ◽

1996 ◽

Vol 29 (5) ◽

pp. 483-489

Author(s):

Lilian Terezinha de Queiroz Leite ◽

Mauricio Resende ◽

Wanderley de Souza ◽

Elizabeth R.S. Camargos ◽

Matilde Cota Koury

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Western Blot ◽

Silver Staining ◽

Colloidal Gold ◽

Leptospira Interrogans ◽

Outer Envelope ◽

Lethal Challenge ◽

Sds Page

Monoclonal antibodies (MABs) ivere produced against an etbylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

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Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.2.2418100 ◽

1986 ◽

Vol 34 (2) ◽

pp. 209-214 ◽

Cited By ~ 40

Author(s):

J U Alles ◽

K Bosslet

Keyword(s):

Molecular Weight ◽

Monoclonal Antibody ◽

Endothelial Cells ◽

Endothelial Cell ◽

Specific Antigen ◽

Similar Molecular Weight ◽

Immunochemical Characterization ◽

Sds Page

A new monoclonal antibody (moab BW 200) of IgG3 kappa-isotype was generated which recognizes an epitope located on an antigen molecule restricted to human neoplastic and non-neoplastic endothelial cells. The molecular weight of the antigen was determined using immunoprecipitation analysis followed by SDS-PAGE. Despite its similar molecular weight to FVIII-RAG, the antigen detected by moab BW 200 was shown to be different from FVIII-RAG.

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Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

Author(s):

Katsuichi Saito ◽

Kazuya Kondo ◽

Ichiro Kojima ◽

Atsushi Yokota ◽

Fusao Tomita

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Extracellular Enzyme ◽

Molecular Weights ◽

Sds Page ◽

Monomeric Structure ◽

Ph Range ◽

Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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