MIGRATION OF SIALIC ACID-RICH PROTEINS ON SDS-PAGE IS SLOWER THAN PREDICTED FROM ACTUAL MOLECULAR WEIGHTS

Author(s):  
Chunlin Qin ◽  
Jan C. Brunn ◽  
William T. Butler
Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1244
Author(s):  
Malik Adil Nawaz ◽  
Tanoj Kumar Singh ◽  
Regine Stockmann ◽  
Hema Jegasothy ◽  
Roman Buckow

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.


1980 ◽  
Vol 30 (2) ◽  
pp. 353-361
Author(s):  
L R Glasgow ◽  
R L Hill

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.


2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita

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 ).


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhengliang L. Wu ◽  
James M. Ertelt

AbstractGlycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.


2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Aleksandra M. Torbica ◽  
Jasna S. Mastilović ◽  
Milica M. Pojić ◽  
Žarko S. Kevrešan

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.


1989 ◽  
Vol 93 (1) ◽  
pp. 123-131
Author(s):  
NANCY J. LANE ◽  
STEPHEN M. DILWORTH

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.


Author(s):  
J. Martinez ◽  
J.E. Palascak

Human fibrinogen is a glycoprotein which contains 6 sialic acid residues per molecule. Enzymatic removal of sialic acid modifies its functional properties as indicated by shortening of the thrombin time due to enhanced asialofibrin monomer polymerization. The abnormal fibrinogen of liver disease contains an Increased amount of sialic acid and is functionally characterized by impaired fibrin monomer polymerization. The prolongation of its thrombin time correlates with its increased sialic acid content. Enzymatic cleavage of the excess sialic acid results in normalization of the thrombin time and the fibrin monomer polymerization. Quantitative labelling of sialic acid with (3H) demonstrates increased labelling of the abnormal fibrinogen compared to normal fibrinogen reflecting the sialic acid content of the abnormal molecule. The radioactivity of the labelled normal and abnormal fibrinogens after reduction and SDS-PAGE was limited to the Bβ and γ chains with 60% of the radioactivity in the Bβ chain and 40% in the γ chain. A similar distribution of radioactivity was found after removal of the excess sialic acid from the respective chains. β-galactose was also increased in the abnormal fibrinogen and paralleled the increase in sialic acid. These studies indicate that sialic acid is distributed normally on the chains of the abnormal fibrinogen of liver disease, but its increased content is responsible for the functional defect of the protein.


1987 ◽  
Author(s):  
N A Booth ◽  
A Reith ◽  
B Bennett

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.


1997 ◽  
Vol 87 (2) ◽  
pp. 197-202 ◽  
Author(s):  
R. Rosario-Cruz ◽  
E. Miranda-Miranda ◽  
Z. Garcia-Vasquez ◽  
M. Ortiz-Estrada

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.


1987 ◽  
Vol 33 (8) ◽  
pp. 698-703 ◽  
Author(s):  
C. P. Kubicek ◽  
T. Panda ◽  
G. Schreferl-kunar ◽  
F. Gruber ◽  
R. Messner

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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