scholarly journals The quaternary structure of wheat-germ aspartate transcarbamoylase

1982 ◽  
Vol 203 (2) ◽  
pp. 413-417 ◽  
Author(s):  
R J Yon ◽  
J E Grayson ◽  
A Chawda ◽  
P J Butterworth
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Quaternary Structure ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Aspartate Transcarbamoylase ◽  
Molecular Masses

1. The molecular mass of aspartate transcarbamoylase purified from wheat germ was found to be 101kDa by sucrose-density-gradient centrifugation, 103kDa by gel-filtration chromatography and 108kDa by polyacrylamide-gel electrophoresis. A mean value of 104 +/- 11kDa was obtained by pooling several replicate results from each method. 2. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated a single size of polypeptide chain of mean molecular mass 37 +/- 4kDa. The ratio of the mean molecular masses of the active and denatured enzymes is 2.8.3. When the active enzyme was covalently cross-linked at a low protein concentration by dimethyl suberimidate, and then examined electrophoretically under denaturing conditions, three size species were observed to predominate, of apparent molecular masses 36, 77 and 106kDa respectively. 4. These results indicate that the intact, fully regulatory enzyme is a simple trimer, slightly larger than the trimeric ‘catalytic subunit’ of the aspartate transcarbamoylase from Escherichia coli [Weber (1968) Nature (London) 218, 1116-1118]. The prevalence of trimeric structures amongst carbamoyl-transferase enzymes is discussed.

scholarly journals Lupus autoantibodies target ribosomal P proteins.

1985 ◽  
Vol 162 (2) ◽  
pp. 459-471 ◽  
Author(s):  
K B Elkon ◽  
A P Parnassa ◽  
C L Foster
Keyword(s):  
Western Blotting ◽  
Lupus Erythematosus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ribosomal Subunit ◽  
Polyacrylamide Gel ◽  
Antibody Activity ◽  
Composite Gel ◽  
Molecular Masses

All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.

scholarly journals Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

2020 ◽  
Author(s):  
Rômulo Leão Silva Neris ◽  
Ajuni Kaur ◽  
Aldrin V. Gomes
Keyword(s):  
Molecular Weight ◽  
Molecular Mass ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Molecular Weights ◽  
Molecular Masses ◽  
Protein Molecular Weight

ABSTRACTThe most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s). We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.

scholarly journals An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88° C

1987 ◽  
Vol 247 (1) ◽  
pp. 121-133 ◽  
Author(s):  
D A Cowan ◽  
K A Smolenski ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Polyacrylamide Gel ◽  
British Library ◽  
Extracellular Proteinase ◽  
Hydrophobic Residues ◽  
The Stability

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

scholarly journals Presence of a high-molecular-weight form of catalase in enzyme purified from mouse liver

1977 ◽  
Vol 163 (3) ◽  
pp. 449-453 ◽  
Author(s):  
M B Baird ◽  
H R Massie ◽  
L S Birnbaum
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
High Molecular Weight Form ◽  
High Molecular Weight Material ◽  
Molecular Weight Form

Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of the total material consists of a form with a higher molecular weight than the bulk of the catalase. The two components were separated by sucrose-gradient centrifugation. Polyacrylamide-gel electrophoresis (in borate buffer) demonstrated that high-molecular-weight catalase is enriched in a more slowly migrating component, and sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the molecular weight of the subunits of the high-molecular-weight material is identical with that of the subunits of the major form. These results suggest that high-molecular-weight catalase consists of subunits that are not markedly distinct from those present in the normal catalase tetramer.

Analysis for Cerebrospinal Fluid Proteins by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

1992 ◽  
Vol 38 (10) ◽  
pp. 2008-2012 ◽  
Author(s):  
F Mashige ◽  
T Shimizu ◽  
S Iijima ◽  
A Ohkubo
Keyword(s):  
Cerebrospinal Fluid ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Apolipoprotein A ◽  
Csf Proteins ◽  
Sds Page ◽  
Molecular Masses

Abstract Cerebrospinal fluid (CSF) proteins with molecular masses of < 150,000 Da were identified by immunoblotting after two kinds of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With PAGE 1 (17-27% gradient gel), CSF proteins were clearly separated into seven to nine bands with molecular masses of 3000-67,000 Da; seven bands were identified as beta 2-microglobulin, lysozyme, prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, and albumin by immunoblotting. With PAGE 2 (10-20% gradient gel), proteins were clearly separated into 11-16 bands with molecular masses of 15,000-150,000 Da; 11 were identified as prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, albumin, alpha 1-antitrypsin, transferrin (separated into two bands), immunoglobulin fragments, haptoglobin, and IgG. We analyzed CSF samples collected from 81 patients with cerebrospinal signs by these SDS-PAGE methods and observed prominent bands in some cases.

scholarly journals Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae

1998 ◽  
Vol 64 (10) ◽  
pp. 3607-3614 ◽  
Author(s):  
Christine Riou ◽  
Jean-Michel Salmon ◽  
Marie-Jose Vallier ◽  
Ziya Günata ◽  
Pierre Barre
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Glucose Tolerant

ABSTRACT Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1mg of protein−1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki , 1.36 M) or glucono-δ-lactone (Ki , 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

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