The Effect of Leucocyte Elastase on the Immunoelectrophoretic Behaviour of α1-Antitrypsin

1984 ◽  
Vol 66 (2) ◽  
pp. 217-224 ◽  
Author(s):  
R. A. Stockley ◽  
S. C. Afford
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Enzyme Inhibitor ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Two Dimensional ◽  
Ph Values ◽  
Inhibitory Capacity ◽  
Leucocyte Elastase ◽  
Enzyme Complexes

1. Two-dimensional immunoelectrophoresis and conventional sodium dodecyl sulphate-polyacryl-amide gel electrophoresis was performed on various mixtures of purified α1-antitrypsin (α1AT) and leucocyte elastase (LE). 2. The results confirm that α1AT inhibits LE by the formation of enzyme-inhibitor complexes demonstrable by both techniques. 3. The complexes break down with time and are not affected by pH in the presence of excess α1AT. However, the breakdown is more rapid in the presence of excess enzyme only at pH values where LE remains active. The resultant products of the complex breakdown include inactivated LE and α1AT that has undergone limited proteolysis. 4. It is concluded that the presence or absence of α1AT-enzyme complexes as demonstrated by two-dimensional immunoelectrophoresis must be interpreted with caution when studying α1AT function in lung secretions. The absence of such complexes does not mean that previous interaction with enzyme has not occurred, thereby accounting for a reduction in α1AT inhibitory capacity.

scholarly journals Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

1978 ◽  
Vol 173 (2) ◽  
pp. 553-563 ◽  
Author(s):  
P R Flanagan ◽  
G G Forstner
Keyword(s):  
New Species ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Ph ◽  
Precipitin Line ◽  
Molecular Weights ◽  
Ph Values

Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10–20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3–6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.

Irreversible heat denaturation of bovine α-lactalbumin

1986 ◽  
Vol 53 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Lesley C. Chaplin ◽  
Richard L. J. Lyster
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Denaturation ◽  
Two Dimensional ◽  
Native Protein ◽  
Denatured Protein ◽  
Hydrolysis Of

SUMMARYThe irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.

Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Parasitology ◽  
1989 ◽  
Vol 99 (2) ◽  
pp. 175-187 ◽  
Author(s):  
C. A. Sutton ◽  
M. W. Shirley ◽  
M. H. Wisher
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Sds Page

SummaryTwo dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS–PAGE) has been used to produce ‘fingerprint’ maps of the proteins from each of the 7 species of Eimeria which infect the chicken. All 7 species could be identified from their array of polypeptides but few differences were detected between strains of the same species. Alterations to the polypeptide array associated with the stage of sporulation of the oocysts were observed. lodination of sporozoites, 2D SDS–PAGE, autoradiography and immunoblotting techniques were combined to identify polypeptides with a surface moiety and those which were antigenic.

scholarly journals Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats

1983 ◽  
Vol 215 (1) ◽  
pp. 83-89 ◽  
Author(s):  
H Sakai ◽  
Y Hino ◽  
S Minakami
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Liver Microsomes ◽  
Polyacrylamide Gel ◽  
Tryptic Digestion ◽  
Post Translational Modification ◽  
Cytochrome P 450

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

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