scholarly journals Structural studies on the glycoproteins from bovine cervical mucus

1978 ◽  
Vol 173 (3) ◽  
pp. 941-947 ◽  
Author(s):  
G P Roberts
Keyword(s):  
Physical Properties ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Cervical Mucus ◽  
Cysteine Residues ◽  
Structural Studies ◽  
Disulphide Bonds ◽  
Structural Differences ◽  
Sepharose 4B

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely ‘naked’ peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.

scholarly journals Isolation, characterization and synthesis of α-foetoprotein from neonatal-rat skin

1983 ◽  
Vol 216 (1) ◽  
pp. 227-231
Author(s):  
K Mujoo ◽  
M Ali ◽  
M K Sahib
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Explant Culture ◽  
Polyacrylamide Gel ◽  
Neonatal Rat ◽  
Rat Skin ◽  
Packed Volume ◽  
Sepharose 4B

Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 × 10(9)M-1 by dextran-coated-charcoal and 0.75 × 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.

scholarly journals Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

1986 ◽  
Vol 234 (1) ◽  
pp. 157-162 ◽  
Author(s):  
N N Dewji ◽  
D R De-Keyzer ◽  
J L Stirling
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Alpha Subunit ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

scholarly journals Purification of an exo-β-D-glucanase from cell-free extracts of Candida utilis

1976 ◽  
Vol 159 (3) ◽  
pp. 555-562 ◽  
Author(s):  
V Notario ◽  
T G Villa ◽  
J R Villanueva
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Acidic Amino Acids ◽  
Sepharose 4B ◽  
Hydrolysis Of

β-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific β-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-β-linkages by an exo-splitting mechanism. Glucono-δ-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.

scholarly journals Further characterization of the cold agglutinin from the snail Achatina fulica

1987 ◽  
Vol 242 (2) ◽  
pp. 331-338 ◽  
Author(s):  
D Mitra ◽  
M Sarkar ◽  
A K Allen
Keyword(s):  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Random Coil ◽  
Cold Agglutinin ◽  
Affinity Column ◽  
Human Umbilical Cord ◽  
Achatina Fulica ◽  
Sepharose 4B ◽  
Albumin Gland

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Identification of novel α-gliadin genes

Genome ◽  
2011 ◽  
Vol 54 (3) ◽  
pp. 244-252 ◽  
Author(s):  
Peng-Fei Qi ◽  
Yu-Ming Wei ◽  
Qing Chen ◽  
Thérèse Ouellet ◽  
Jia Ai ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Triticum Aestivum L ◽  
Protein Band ◽  
Cysteine Residues ◽  
Disulphide Bonds ◽  
Chain Extenders ◽  
Domain I ◽  
Unique Domain

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat ( Triticum aestivum L.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS–PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni

1988 ◽  
Vol 254 (2) ◽  
pp. 419-426 ◽  
Author(s):  
P M Wiest ◽  
E J Tisdale ◽  
W L Roberts ◽  
T L Rosenberry ◽  
A A F Mahmoud ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Free Amino ◽  
Protein Modification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Reductive Methylation ◽  
Amide Linkage

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

Sign in / Sign up

Export Citation Format

Share Document