scholarly journals More Than One Disease Process in Chronic Sinusitis Based on Mucin Fragmentation Patterns and Amino Acid Analysis

2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Mahmoud El-Sayed Ali ◽  
Jeffrey P. Pearson
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Chronic Sinusitis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disease Process ◽  
Gel Chromatography ◽  
Sds Page ◽  
Amino Acid Contents ◽  
Fragmentation Patterns

Objective. To characterise fragmentation patterns and amino acid composition of MUC2 and MUC5AC in chronic sinusitis.Methods. Antigenic identity of purified sinus mucins was determined by ELISA. Fragmentation patterns of a MUC5AC rich sample mucin were analysed by Sepharose CL-2B gel chromatography. Samples, divided into one MUC2 rich and one MUC5AC rich group, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their amino acid contents were analysed.Results. Reduction, trypsin digestion, and papain digestion produced progressively smaller mucin species. On SDS-PAGE, digested MUC5AC rich mucin produced four distinct products. Amino acid analysis was characteristic of mucins with high serine, threonine, and proline contents and reduction and proteolysis increased relative proportions of these amino acids. MUC5AC rich mucins contained more protein than MUC2 rich mucins.Conclusion. Sinus mucin fragmentation produced mucin subunits and glycopeptide units of smaller molecular sizes which are likely to have lower viscoelastic properties. Applying this in vivo could alter mucus physical properties and biologic functions. Amino acid contents of MUC2 and MUC5AC mucins are different. This could be contributing to biological properties and functions of sinus mucins. These data suggest that there may be different pathological processes occurring at the cellular level on chronic sinusitis.

scholarly journals Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 518
Author(s):  
Honghai Zhang ◽  
Yunpeng Zhang ◽  
Tie Yin ◽  
Jing Wang ◽  
Xiaolin Zhang
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Degrading Enzyme ◽  
Sds Page ◽  
Ochratoxin Α ◽  
Acid Amidohydrolase

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Abstract Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

1993 ◽  
Vol 265 (5) ◽  
pp. G894-G902 ◽  
Author(s):  
D. D. Stump ◽  
S. L. Zhou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Binding ◽  
Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies

Genome ◽  
2006 ◽  
Vol 49 (2) ◽  
pp. 181-189 ◽  
Author(s):  
H Q Wang ◽  
X Y Zhang
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Storage Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutenin Subunits ◽  
Sds Page

High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives.Key words: wheat, HMW-GS, monoclonal antibody, immunoscreen.

scholarly journals Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

2002 ◽  
Vol 68 (7) ◽  
pp. 3532-3536 ◽  
Author(s):  
María J. Benito ◽  
Mar Rodríguez ◽  
Félix Núñez ◽  
Miguel A. Asensio ◽  
María E. Bermúdez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Penicillium Chrysogenum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Extracellular Protease ◽  
Collagenolytic Activity ◽  
Sds Page

ABSTRACT An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

scholarly journals Purification of human erythropoietin to homogeneity by a rapid five- step procedure

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 71-79 ◽  
Author(s):  
G Krystal ◽  
HR Pankratz ◽  
NM Farber ◽  
JE Smart
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Assay ◽  
Tween 20 ◽  
Reverse Phase ◽  
Vitro Assay ◽  
Step Procedure ◽  
Sds Page

Abstract Human urinary erythropoietin (Ep) has been purified using a simple five- step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

scholarly journals Purification of human erythropoietin to homogeneity by a rapid five- step procedure

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 71-79 ◽  
Author(s):  
G Krystal ◽  
HR Pankratz ◽  
NM Farber ◽  
JE Smart
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Assay ◽  
Tween 20 ◽  
Reverse Phase ◽  
Vitro Assay ◽  
Step Procedure ◽  
Sds Page

Human urinary erythropoietin (Ep) has been purified using a simple five- step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

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