An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

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Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

Biochemical Journal ◽

10.1042/bj3250761 ◽

1997 ◽

Vol 325 (3) ◽

pp. 761-769 ◽

Cited By ~ 64

Author(s):

Isabelle GARCIA ◽

Matthew RODGERS ◽

Catherine LENNE ◽

Anne ROLLAND ◽

Alain SAILLAND ◽

...

Keyword(s):

Nucleotide Sequence ◽

Gel Filtration ◽

Peptide Sequence ◽

Amino Acid Residues ◽

Carrot Cells ◽

Expression Studies ◽

Sds Page ◽

Molecular Masses ◽

The Difference ◽

Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

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Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates

Foods ◽

10.3390/foods10061244 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1244

Author(s):

Malik Adil Nawaz ◽

Tanoj Kumar Singh ◽

Regine Stockmann ◽

Hema Jegasothy ◽

Roman Buckow

Keyword(s):

Physicochemical Properties ◽

Faba Bean ◽

Protein Concentration ◽

High Concentration ◽

Molecular Weights ◽

Oil In Water ◽

Sds Page ◽

Lower Protein ◽

7S Globulin ◽

Bean Protein

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~β-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5–8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5–6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7–8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.

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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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Effects of Wheat Bug (Eurygasterspp. andAeliaspp.) Infestation in Preharvest Period on Wheat Technological Quality and Gluten Composition

The Scientific World JOURNAL ◽

10.1155/2014/148025 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Aleksandra M. Torbica ◽

Jasna S. Mastilović ◽

Milica M. Pojić ◽

Žarko S. Kevrešan

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Wheat Gluten ◽

Sodium Dodecyl ◽

Deformation Energy ◽

Wheat Varieties ◽

Gluten Proteins ◽

Molecular Weights ◽

Sds Page ◽

Technological Quality ◽

Gluten Index

The effects of wheat bug infestation (Eurygasterspp. andAeliaspp.) on the composition of wheat gluten proteins and its influence on flour technological quality were investigated in the present study. Wheat samples of six wheat varieties, collected from two localities in northern Serbia, were characterized by significantly different level of wheat bug infestation. Composition of wheat gluten proteins was determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), while the selected parameters of technological quality were determined according to standard and modified empirical rheological methods (Farinograph, Extensograph, Alveograph, and Gluten Index). The surface morphology of the selected samples was viewed using scanning electron microscopy (SEM). Wheat from wheat bug-infested locality regardless of the variety had deteriorated technological quality expressed with higher Farinograph softening degree, lower or immeasurable Extensograph energy, and Alveograph deformation energy. The most important changes in the gluten proteins composition of bug-infested wheat were related to gliadin subunits with molecular weights below 75 kDa, which consequently caused deterioration of uniaxial and biaxial extensibility and dough softening during mixing.

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

Journal of Cell Science ◽

10.1242/jcs.93.1.123 ◽

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.

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A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY

10.1055/s-0038-1644459 ◽

1987 ◽

Author(s):

N A Booth ◽

A Reith ◽

B Bennett

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Plasminogen Activator ◽

Apparent Molecular Weight ◽

Late Pregnancy ◽

Plasminogen Activator Inhibitor ◽

Molecular Weights ◽

Histiocytic Cell ◽

Sds Page ◽

Pai 1

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.

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Construction and Characterization of anagr Deletion Mutant of Staphylococcus epidermidis

Infection and Immunity ◽

10.1128/iai.68.3.1048-1053.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1048-1053 ◽

Cited By ~ 90

Author(s):

Cuong Vuong ◽

Friedrich Götz ◽

Michael Otto

Keyword(s):

Staphylococcus Epidermidis ◽

Deletion Mutant ◽

Protein Pattern ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Surface Proteins ◽

Accessory Gene ◽

Accessory Gene Regulator ◽

Sds Page ◽

The Difference

ABSTRACT The physiological significance of the accessory gene regulator (agr) system of Staphylococcus epidermidis was investigated by construction of an agr deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The agrsystem of S. epidermidis thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for Staphylococcus aureus. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the agr mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the agr mutant.

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Detection of esterase activity in susceptible and organophosphate resistant strains of the cattle tick Boophilus microplus (Acari: Ixodidae)

Bulletin of Entomological Research ◽

10.1017/s0007485300027358 ◽

1997 ◽

Vol 87 (2) ◽

pp. 197-202 ◽

Cited By ~ 26

Author(s):

R. Rosario-Cruz ◽

E. Miranda-Miranda ◽

Z. Garcia-Vasquez ◽

M. Ortiz-Estrada

Keyword(s):

Esterase Activity ◽

Boophilus Microplus ◽

Cattle Tick ◽

Molecular Weights ◽

Sds Page ◽

Genes Encoding ◽

Resistant Strains ◽

Hydrolysis Of ◽

Coomassie Brilliant ◽

Naphthyl Acetate

AbstractTwo organophosphate (OP) resistant strains of the cattle tick Boophilus microplus (Canestrini) from Mexico and Costa Rica were used to analyse the presence of esterase activity associated with resistance. The concentrations of six major proteins in both resistant strains were increased compared to the susceptible Morelos strain, both when stained with Coomassie Brilliant Blue after SDS-PAGE, and when analysed for esterase activity by the hydrolysis of naphthyl acetate esters. Esterases were named A or B in relation to the substrate preference for alpha or beta naphthyl acetate and numbered according to their position on the SDS—PAGE. The molecular weights of these proteins were: 125, 115, 108, 77, 43 and 67 Kd for Est-Bl, Est-B2, Est-B3, Est-B4, Est-B5 and Est-A respectively. Est-B3 showed cholinesterase (ChE) activity. This study strengthens the hypothesis that the mechanism associated with OP resistance found in many other insects includes an increase of esterase activity, probably as a result of gene amplification. The genes encoding these enzymes could be potentially used as molecular markers to detect resistance in the cattle tick B. microplus using a DNA probe.

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O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei

Canadian Journal of Microbiology ◽

10.1139/m87-122 ◽

1987 ◽

Vol 33 (8) ◽

pp. 698-703 ◽

Cited By ~ 51

Author(s):

C. P. Kubicek ◽

T. Panda ◽

G. Schreferl-kunar ◽

F. Gruber ◽

R. Messner

Keyword(s):

Trichoderma Reesei ◽

Protein Glycosylation ◽

The Other ◽

Detectable Effect ◽

Secreted Protein ◽

Intracellular Protein ◽

Molecular Weights ◽

Sds Page ◽

Wide Range ◽

Molecular Masses

The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.

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