An electrophoretic characterization of iron-transporting proteins in Mannheimia haemolytica A1

2013 ◽  
Vol 16 (3) ◽  
pp. 527-532 ◽  
Author(s):  
A. Puchalski ◽  
R. Urban-Chmiel ◽  
M. Dec ◽  
A. Wernicki
Keyword(s):  
Immune Response ◽  
Iron Uptake ◽  
Outer Membrane Proteins ◽  
Mannheimia Haemolytica ◽  
Iron Ions ◽  
Growth Environment ◽  
Molecular Weights ◽  
Sds Page ◽  
Isoelectric Points

AbstractIron-regulated outer membrane proteins (IROMPs) in Mannheimia haemolytica A1, which function as a receptor for complexes containing iron ions, are induced by iron deficiency in the growth environment of the bacteria. Densitometric analysis of SDS-PAGE separation showed expression of IROMPs of 71, 77, and 100 kDa in the case of bacteria grown in a medium with 2,2-dipyridyl. The electrophoregrams obtained in 2-DE separations confirmed the presence of protein fractions with these molecular weights and isoelectric points ranging from 5.4 to 6.4. The results of the study also confirmed the ability of M. haemolytica A1 proteins involved in iron uptake to induce a protective immune response. In Western blot with serum from convalescent calves naturally infected with M. haemolytica A1, distinct reactions were obtained for IROMPs of 71, 77, and 100 kDa.

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 ).

Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties

1989 ◽  
Vol 93 (1) ◽  
pp. 123-131
Author(s):  
NANCY J. LANE ◽  
STEPHEN M. DILWORTH
Keyword(s):  
Periplaneta Americana ◽  
Biochemical Characterization ◽  
Septate Junction ◽  
Septate Junctions ◽  
Molecular Weights ◽  
Thin Sectioning ◽  
Sds Page ◽  
High Concentrations ◽  
Membrane Components

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.

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 Inhibin from Culture and Non Culture of Granulose Cells for Monoclonal Antibody of Inhibin Production

2010 ◽  
Vol 4 (1) ◽  
Author(s):  
Amiruddin Amiruddin ◽  
Tongku Nizwan Siregar ◽  
Amalia Sutriana ◽  
Dwinna Aliza ◽  
T. Armansyah
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Western Blot ◽  
Total Amino Acid ◽  
Multiple Ovulation ◽  
Sds Page ◽  
Isoelectric Points ◽  
Granulose Cells

This study has long-term objectives to obtain immunogenic prototype that can be used to induce multiple ovulation in goats. Working steps of this study were begun with the collection of ovarium from goats, collection of granulose cells, culture of granulose and characterization of molecular weight and isoelectric point (pI) of inhibin protein of granulose cells obtained from culture and non-culture of granulose cells, and followed by preparation of monoclonal antibody toward inhibin. The results showed that inhibin isolated either from culture or non-culture of granulose cells produced a 32 kDa band. Molecular weight of inhibin was measured by Western Blot. The 32 kDa band of SDS PAGE product appeared on Western Blot result was inhibin molecules produced by granulose cells collected fom culture and non-culture of granulose cells that can be identified by Mab-inhibin. Product of IEF gel electrophoresis suggested that inhibin molecule collected from culture of granulose cells has no charge at isoelectric points ranging from 5-6, depends on its total amino acid composition.

Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

2018 ◽  
Author(s):  
Losa Rose ◽  
Bablu Kumar ◽  
Ankita Jain ◽  
M K Singh ◽  
Abhishek .
Keyword(s):  
Immune Response ◽  
Outer Membrane ◽  
Brucella Abortus ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Biologically Active ◽  
Cell Mediated Immunity ◽  
Humoral Immune ◽  
Sds Page

Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

scholarly journals Protein and antigen profiles of Leptospira interrogans serovar Hardjo

2009 ◽  
Vol 39 (9) ◽  
pp. 2539-2543
Author(s):  
Bárbara Nobre Lafetá ◽  
Elaine Cristina de Castro ◽  
Nivaldo da Silva
Keyword(s):  
Immune Response ◽  
Membrane Proteins ◽  
Western Blot ◽  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Rabbit Antiserum ◽  
Leptospira Interrogans ◽  
Sds Page ◽  
Hyperimmune Rabbit

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

2013 ◽  
Vol 8 (12) ◽  
pp. 1183-1193 ◽  
Author(s):  
Marcin Maciąga ◽  
Michał Szkop ◽  
Andrzej Paszkowski
Keyword(s):  
Common Wheat ◽  
Aspartate Aminotransferase ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Amino Acid Sequences ◽  
Molecular Weights ◽  
Sds Page ◽  
Technique Comparison

AbstractSix allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

scholarly journals Purification and Characterization of Strong Simultaneous Enzyme Production of Protease and α-Amylase from an Extremophile-Bacillus sp. FW2 and Its Possibility in Food Waste Degradation

Fermentation ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. 12
Author(s):  
Van Hong Thi Pham ◽  
Jaisoo Kim ◽  
Jeahong Shim ◽  
Soonwoong Chang ◽  
Woojin Chung
Keyword(s):  
Food Waste ◽  
Environmental Conditions ◽  
Enzyme Production ◽  
Molecular Weights ◽  
Sds Page ◽  
Metabolic Products ◽  
Wide Ph Range ◽  
Waste Degradation ◽  
Ph Range

Microbial enzymes such as protease and amylase are valuable enzymes with various applications, widely investigated for their applications in degradation of organic waste, biofuel industries, agricultural, pharmaceuticals, chemistry, and biotechnology. In particular, extremophiles play an important role in biorefinery due to their novel metabolic products such as high value catalytic enzymes that are active even under harsh environmental conditions. Due to their potentials and very broad activities, this study isolated, investigated, and characterized the protease- and amylase-producing bacterial strain FW2 that was isolated from food waste. Strain FW2 belongs to the genus Bacillus and was found to be closest to Bacillus amyloliquefaciens DSM 7T with a similarity of 99.86%. This strain was able to degrade organic compounds at temperatures from −6 °C to 75 °C (but weak at 80 °C) under a wide pH range (4.5–12) and high-salinity conditions up to 35% NaCl. Maximum enzyme production was obtained at 1200 ± 23.4 U/mL for protease and 2400 ± 45.8 U/mL for amylase for 4 days at pH 7–7.5, 40–45 °C, and 0–10% NaCl. SDS-PAGE analysis showed that the molecular weights of purified protease were 28 kDa and 44 kDa, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively, and molecular weight of α-amylase was 55 kDa. Degradation food waste was determined after 15 days, observing a 69% of volume decrease. A potential commercial extremozyme-producing bacteria such as strain FW2 may be a promising contributor to waste degradation under extreme environmental conditions.

Characterization of Plastid 5-Aminolevulinate Dehydratase (ALAD; EC 4.2.1.24) from Spinach (Spinacia olevacea L.) by Sequencing and Comparison with Non-Plant ALAD Enzymes

1992 ◽  
Vol 47 (1-2) ◽  
pp. 77-84 ◽  
Author(s):  
Anke Schaumburg ◽  
Hansjörg A. W. Schneider-Poetsch ◽  
C. Eckerskorn
Keyword(s):  
Amino Acids ◽  
Posttranslational Modifications ◽  
Biochemical Properties ◽  
Amino Acid Residues ◽  
Active Centre ◽  
Molecular Weights ◽  
Sds Page ◽  
Aminolevulinate Dehydratase ◽  
Plant Enzyme

Abstract We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant. A fulllength cDNA clone (1727 bp) encoding this enzyme has been identified by immunoscreening a lambda gt 11 cDNA library of spinach. ALAD is not a plant-specific enzyme; however, the plant enzyme differs from the well known ALAD enzymes of bacteria, yeast and animals in structural and biochemical properties and in that it is located in the plastid. Differences and homologies can be traced back to the molecular level. The mature ALAD subunit, whose N-terminus was determined by automatic Edman degradation, is a protein of 367 amino acid residues and has a Mr of 40,132. This figure is in the range of molecular weights of non-plant ALADs. The active centre is highly conserved and the same is true for the ion-binding domain, except that 4 cysteines of the non-plant enzymes (binding Zn2+) have disappeared and a total of 6 aspartic acids meets the demands of Mg2+-binding. However, there are more distinct differences. Apart from a transit sequence of 56 amino acids targeting the plastid, the N-terminal part of the mature plant enzyme differs considerably from non-plant ALAD enzymes. It is rich in prolines and hydroxylated amino acids. The apparent Mr on SDS-PAGE is 45,000 or higher, but up to now posttranslational modifications have not been found.

scholarly journals Comparison of pectolytic and proteolytic activities of six clarifying commercial preparations. Incidence of secondary enzymatic activities on proteins in Champagne musts

OENO One ◽  
1996 ◽  
Vol 30 (2) ◽  
pp. 103
Author(s):  
Laurent Berthier ◽  
Richard Marchal ◽  
Alain Maujean
Keyword(s):  
Isoelectric Focusing ◽  
Total Protein ◽  
Protein Concentration ◽  
Enzymatic Activities ◽  
Pinot Noir ◽  
Molecular Weights ◽  
Sds Page ◽  
Isoelectric Points ◽  
Proteolytic Activities ◽  
Grape Varieties

<p style="text-align: justify;">In order ta estimate c1arifying aptitude and the effects of prefermentation treatment by commercial pectinases, polygalacturonase (PG), pectin Iyase (PL), pectinmethylesterase (PME) and proteolytic activities of six pectinases samples were studied. PG activity is preponderate and proportional to the relative protein concentration.</p><p style="text-align: justify;">These one, submitted to both SDS-PAGE and isoelectric focusing technics, show molecular weights ranging from 20 ta 100 kDa and isoelectric points ranging from 3.00 ta 7.40. Total protein of Champagne grape varieties Chardonnay (CH) and Pinot noir (PN) - were submitted to an isoelectric focusing. All of the pI's ranging from 3.30 to 4.55 (CH) and from 3.30 to 4.90 (PN). Proteases are put in evidence in commercial pectinases using fluorescamine- casein as substrate. However, they seem to have no incidence upon must proteins.</p>

Sign in / Sign up

Export Citation Format

Share Document