scholarly journals Electrophoretic Characterization of Venom Proteins of Montivipera xanthina (Gray, 1849) (Ophidia: Viperidae)

2017 ◽  
Author(s):  
Hüseyin Arıkan ◽  
Nurşen Alpagut Keskin ◽  
Kerim Çiçek
Keyword(s):  
Serine Proteases ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sds Page ◽  
Dependent Activity ◽  
Fibrinogenolytic Activity ◽  
Identification And Characterization ◽  
Insight Into ◽  
Venom Proteins

In this study, with the aim of evaluating coagulant activities in the venom of M. xanthina, we analyzed venom proteins, digestion patterns of fibrinogen chains with venom and the effects of protease inhibitors on M. xanthina venom proteases using Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. Venom samples were obtained from four adult specimens of Montivipera xanthina collected in Gümüldür (Izmir, Turkey). SDS-PAGE analysis demonstrated that 17 protein bands in the range of 20–250 kDa were present. The specific digestion patterns of fibrinogen chains revealed that M. xanthina venom possesses fibrinogenolytic enzymes which could be included in coagulation processes during envenomation Fibrinogenolytic activity directed exclusively towards the Aa-chain with a time-dependent activity towards Bb-chains suggests the presence of both metalloproteinases and serine proteases in M. xanthine venom. In the present study, the occurrence and inhibition of fibrinogenolytic activity of M. xanthina venom were clearly observed. For further analysis, the isolation, identification, and characterization of individual venom components will provide insight into their function and biological roles.

scholarly journals Renaturation of telomere-binding proteins after the fractionation by SDS-polyacrylamide gel electrophoresis

2008 ◽  
Vol 53 (No. 7) ◽  
pp. 317-320
Author(s):  
G. Rotková
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Simple Method ◽  
Telomeric Sequences ◽  
Sds Page ◽  
Identification And Characterization

A simple method for identification and characterization of telomere-binding proteins is described in this article. After Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE), proteins are eluted, renatured and used for retardation analysis with labelled oligonucleotides corresponding to human and plant of telomeric sequences. We show here that this method is efficient to recover sequence-specific DNA-binding abilities of putative telomere-binding proteins.

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

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.

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

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
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 ).

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Identification and characterization of cDNA clones encoding two homologous proteins that are part of the asialoglycoprotein receptor

1987 ◽  
Vol 7 (5) ◽  
pp. 1841-1847
Author(s):  
M McPhaul ◽  
P Berg
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Asialoglycoprotein Receptor ◽  
Cdna Clones ◽  
Differential Splicing ◽  
Homologous Proteins ◽  
Different Types ◽  
Identification And Characterization

The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979

1983 ◽  
Vol 29 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Fraser E. Ashton ◽  
J. Alan Ryan ◽  
Colina Jones ◽  
Bernard R. Brodeur ◽  
Benito B. Diena
Keyword(s):  
Neisseria Meningitidis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Age Groups ◽  
Double Diffusion ◽  
Sds Page ◽  
Class 1 ◽  
Group B

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81 %) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41 500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b,77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS–PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Characterization of metallothionein protein from hepatopancreas organ of Pilsbryoconcha exilis collected from Cikaniki River, Western Java, Indonesia

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
SATA YOSHIDA SRIE RAHAYU ◽  
WAHYU PRIHATINI
Keyword(s):  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
The Body ◽  
Freshwater Environment ◽  
Sds Page ◽  
Freshwater Biota ◽  
Aas Method ◽  
Endogenous Proteins

Abstract. Rahayu SYS, Prihatini W. 2020. Characterization of metallothionein protein from hepatopancreas organ of Pilsbryoconcha exilis collected from Cikaniki River, Western Java, Indonesia. Nusantara Bioscience 12: 1-5. Freshwater environment, undergoing various changes due to the presence of dangerous toxic anthropogenic waste. It causes pressure on the freshwater biota that lives in it, such as Pilsbryoconcha exilis mussel at the bottom of freshwater. This pressure is controlled by the body through the synthesis of a set of stress proteins. Endogenous proteins, metallothionein (MT), in the body of freshwater biota absorb heavy metals in the body of biota, in the form of stress control. This research identified MT protein on P. exilis from contaminated waters such as the Cikaniki river with the average of mercury levels in water, sediment, and hepatopancreas of mussels using AAS method were 0.001 mg/L, 0.120 mg/L, and 1.318 mg/L respectively. Hepatopancreas of P. exilis was extracted using a Tissue Extraction Reagent I kit (Invitrogen), with procedures following the factory manual. The extract was purified by filtration using Sephadec 50; then, the filtration results were migrated together with the PageRuler TM Unstained Low Range Protein Ladder (Fermentas) in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) gel medium on Biorad Protein II electrophoresis. After completion of electrophoresis, the gel was stained using Page Blue Protein Staining Solution (Fermentas), following the factory manual procedure. Characterization at this research has succeeded in obtaining the MT-I isoform protein measuring 5, 10, and 25 kDa from the hepatopancreas organ of P. exilis.

Identification and characterization of a pancreatic intrinsic factor in the dog

1989 ◽  
Vol 256 (3) ◽  
pp. G517-G523 ◽  
Author(s):  
R. M. Batt ◽  
N. U. Horadagoda ◽  
L. McLean ◽  
D. B. Morton ◽  
K. W. Simpson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Factor ◽  
Gel Filtration ◽  
Polyclonal Antiserum ◽  
Pure Pancreatic Juice ◽  
Intrinsic Factors ◽  
Rabbit Polyclonal Antiserum ◽  
Identification And Characterization

An intrinsic factor has been identified in the canine pancreas, and output and properties of this protein have been compared with those of gastric intrinsic factor in the dog. Mean concentrations of intrinsic factor and peak outputs per minute were approximately 5- to 10-fold higher in pure pancreatic juice after stimulation with secretin and cholecystokinin, respectively, than in pentagastrin-stimulated gastric juice. Purified gastric and pancreatic intrinsic factors had an identical molecular mass of 65 kDa, estimated by gel filtration on Sephacryl S-200, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated single bands corresponding to 53 kDa. Immunoblots showed that rabbit polyclonal antiserum to canine gastric intrinsic factor cross-reacted with canine pancreatic intrinsic factor. Gastric and pancreatic intrinsic factor-cyano[57Co]cobalamin complexes exhibited comparable association constants for ileal receptors in canine brush-border vesicles, while there was minimal binding to jejunal vesicles. These findings demonstrate that the canine pancreas is an important source of an intrinsic factor that closely resembles gastric intrinsic factor in the dog.

scholarly journals Purification and Characterization of an Extracellular Protease from the Fish Pathogen Yersinia ruckeri and Effect of Culture Conditions on Production

1999 ◽  
Vol 65 (9) ◽  
pp. 3969-3975 ◽  
Author(s):  
P. Secades ◽  
J. A. Guijarro
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Exchange Chromatography ◽  
Casamino Acid ◽  
Exponential Growth Phase ◽  
Yersinia Ruckeri ◽  
Fish Pathogen ◽  
Sds Page

ABSTRACT A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogenYersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42°C and had an optimum activity at 37°C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42°C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg2+ or Ca2+ for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. TwoN-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.

Sign in / Sign up

Export Citation Format

Share Document