scholarly journals Structural studies on the major component of Gladiolus style mucilage, an arabinogalactan-protein

1979 ◽  
Vol 181 (3) ◽  
pp. 607-621 ◽  
Author(s):  
P A Gleeson ◽  
A E Clarke
Keyword(s):  
Marked Decrease ◽  
Carbohydrate Antigen ◽  
Arabinogalactan Protein ◽  
Structural Studies ◽  
Tridacna Maxima ◽  
Compatible Pollen ◽  
Sepharose 4B ◽  
Protein Moiety ◽  
Binding Lectin ◽  
Single Symmetrical Peak

The major component of the Gladiolus style mucilage was shown to be an arabinogalactan-protein. The arabinogalactan-protein was isolated from the style extract by affinity chromatography with tridacnin (the galactose-binding lectin from the clam Tridacna maxima) coupled to Sepharose 4B. The isolated arabinogalactan-protein represents 40% of the soluble style extract; it contains 90% (w/w) carbohydrate and 3% protein. The major monosaccharides of the carbohydrate component are galactose and arabinose, in the proportions 6:1. A component with a similar composition was also isolated from the crude extract by precipitation with the beta-glucosyl artifical carbohydrate antigen. The protein moiety of the arabinogalactan-protein remained associated with the carbohydrate after chromatography in urea, and has high contents of serine, glutamic acid, aspartic acid, glycine and alanine. The arabinogalactan-protein is apparently chemically homogeneous; it eluted as a single symmetrical peak from Sepharose 4B, and three fractions collected across the peak were structurally similar. Ultracentrifugal studies showed it to be polydisperse in the mol.wt. range 150 000–400 000. The information obtained from methylation analyses, oxalic acid and enzymic hydrolyses is consistent with a model having a beta 1 leads to 3 galactan backbone, branched through C(O)6 to beta 1 leads to 6 galactan side chains. The arabinose is exclusively present as terminal alpha-L-arabinofuranosyl residues. Enzymic removal of the arabinose residues resulted in a marked decrease in solubility of the molecule. The localization of the arabinogalactan-protein in the mucilage of the style canal was demonstrated cytochemically. The possible roles of the arabinogalactan-protein in relation to recognition of compatible pollen and pollen-tube growth are discussed.

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum Ryegrass Endosperm Cells. I. Hydroxylation of Peptidyl Proline

1981 ◽  
Vol 8 (2) ◽  
pp. 121
Author(s):  
PC Pollard ◽  
GB Fincher
Keyword(s):  
Lolium Multiflorum ◽  
Total Radioactivity ◽  
Ferrous Ion ◽  
Arabinogalactan Protein ◽  
Trichloracetic Acid ◽  
Radioactive Label ◽  
Insoluble Material ◽  
Imino Acid ◽  
Proline Hydroxylation ◽  
Protein Moiety

Suspension-cultured endosperm cells from L. multiflorum secrete into the medium an arabinogalactan-protein in which the protein moiety is rich in hydroxyproline. When cells are grown in the presence of proline labelled with 14C or 3H, the imino acid is rapidly removed from the medium and radio- activity can subsequently be detected in extracellular trichloracetic acid-soluble, ethanol-insoluble material. In this fraction, which contains the arabinogalactan-protein and other polysaccharides, radioactive label is distributed between proline and hydroxyproline. α,α'-Dipyridyl, a chelator of ferrous ion, has no effect on the total radioactivity secreted but markedly alters the distribution of radioactivity in favour of peptidyl proline. Although this inhibition of peptidyl proline hydroxylation can be reversed by ferrous or zinc ions, it is not possible to conclude that ferrous ion, which is required for hydroxylation in other systems, participates specifically in the reaction in ryegrass endosperm cells. Concomitant with the inhibition of proline hydroxylation, α,α'-dipyridyl suppresses the biosynthesis or secretion of extracellular arabinogalactan-protein and arabinoxylan.

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 Improvements on the purification of mannan-binding lectin and demonstration of its Ca2+-independent association with a C1s-like serine protease

1996 ◽  
Vol 319 (2) ◽  
pp. 329-332 ◽  
Author(s):  
Suet Mien TAN ◽  
Maxey C. M. CHUNG ◽  
Oi Lian KON ◽  
Steffen THIEL ◽  
Szu Hee LEE ◽  
...  
Keyword(s):  
Serine Protease ◽  
Gel Filtration ◽  
Classical Pathway ◽  
Form Analysis ◽  
Independent Association ◽  
Elution Method ◽  
Micro Organisms ◽  
Mannan Binding Lectin ◽  
Sepharose 4B ◽  
Binding Lectin

Mannan-binding lectin (MBL), previously called ‘mannan-binding protein’ or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca2+-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.

scholarly journals Crystallization and preliminary structural studies of champedak galactose-binding lectin

2009 ◽  
Vol 65 (9) ◽  
pp. 895-897 ◽  
Author(s):  
Mads Gabrielsen ◽  
Alan Riboldi-Tunnicliffe ◽  
Puteri Shafinaz Abdul-Rahman ◽  
Emida Mohamed ◽  
Wan Izlina Wan Ibrahim ◽  
...  
Keyword(s):  
Structural Studies ◽  
Binding Lectin ◽  
Galactose Binding Lectin

scholarly journals Purification and characterization of a galactan-reactive agglutinin from the clam Tridacna maxima (Röding) and a study of its combining site

1978 ◽  
Vol 175 (2) ◽  
pp. 467-477 ◽  
Author(s):  
B A Baldo ◽  
W H Sawyer ◽  
R V Stick ◽  
G Uhlenbruck
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diffuse Band ◽  
Ph Optimum ◽  
Tridacna Maxima ◽  
Beta Structure ◽  
Two Peaks ◽  
Binding Lectin ◽  
Galactosyl Residues

1. A beta-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polylecyl larch galactan, D-galactosamine coupled to epoxy-activated Sepharose or acid-treated Sepharose. Elution with N-acetyl-D-galactosamine or lactose displaced the bound lectin, which appeared homogeneous by sedimentation analysis. On immunoelectrophoresis at pH8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the alpha-region. 2. On a alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Ioselectric focusing in solution revealed two peaks of pI4.05 and 4.25 and a shoulder, pI4.0, whereas at least three bands close together (pI3.9-4.3) were seen after electrofusing in gel. 3. The agglutinin is a glycoprotein with a mol.wt. of 470300 +/- 20000. Amino acid analysis revealed no methionine and a significant amount of half-cystine residues. 4. Tridacna lectin is a metalloprotein requiring Ca2+ for its haemagglutinating and precipitating activities. 5. In haemagglutination studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing beta-D-galactosyl residues reacted with the lectin to form precipitates both in gel and in solution. Inhibition experiments showed that N-acetyl-D-galactosamine was the best inhibitor of the agglutinin combining sites, followed by p-nitrophenyl beta-D-galactoside, methyl beta-D-galactoside, D-galactosamine and 60O-beta-D-galactopyranosyl-D-galactopyranose. On a molar basis, N-acetyl-D-galactosamine was 20-fold more active than D-galactose and nearly 10-fold more inhibitory than D-galactosamine. 7. Circular-dichroism studies showed that the lectin contains a relatively high proportion of beta-structure. 8. Mercaptoethanol treatment of the agglutinin followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed subunits with approx. mol.wts. of 10000, 20000 and 40000.

scholarly journals Reaction of Some Invertebrate and Plant Agglutinins and a Mouse Myeloma Anti-Galactan Protein With an Arabinogalactan From Wheat

1978 ◽  
Vol 31 (2) ◽  
pp. 149 ◽  
Author(s):  
BA Baldo ◽  
H Neukom ◽  
BA Stone ◽  
G Uhlenbruck
Keyword(s):  
Ricinus Communis ◽  
Lolium Multiflorum ◽  
Arabinogalactan Protein ◽  
Myeloma Protein ◽  
Tridacna Maxima ◽  
The Difference ◽  
Inhibition Studies ◽  
Mild Acid ◽  
Mouse Myeloma ◽  
Galactosyl Residues

Plant, invertebrate and vertebrate proteins which show anti-galactan combining specificities were used in precipitation and inhibition studies with arabinogalactan preparations from wheat and ryegrass (Lolium multiflorum). Of the agglutinins studied, only mouse anti-galactan myeloma protein J539 showed strong reactivity with wheat arabinoglactan-peptide. Weak reactions were observed with the agglutinins from the clam Tridacna maxima, the sponge Axinella polypoides and the anemone Cerianthus membranaceus. No reactions were detected with lectins from the plants Abrus precatorius and Ricinus communis. Reactions readily occurred between Lolium arabinogalactan-protein and the invertebrate and vertebrate agglutinins. Removal of terminal arabinosyl residues from the wheat and Lotium arabinogalactans either by mild acid hydrolysis or by treatment with an arabinofuranosidase increased the reactivity of both peptidoglycans with all of the agglutinins examined except the Ricinus RCAl lectin. Results obtained with wheat arabinogalactan indicate that few D-galactose units are terminal and available for reaction. The difference in reactivities between the wheat and Lotium arabinogalactans may be due to the differences in the galactose:arabinose ratios or to differences in linkage of the galactosyl residues on the two peptidoglycans, or both. Results indicate that the mouse anti-galactan could be a useful reagent for the subcellular localization of wheat arabinogalactan and that tridacnin and Axinella agglutinins could be used to localize the arabinogalactan in L. multiflorum cells.

Purification of Staphylocoagulase by a Bovine Prothrombin-Sepharose 4B Column and its Physicochemical Properties

1979 ◽  
Author(s):  
H. Igarashi ◽  
T. Morita ◽  
S. Iwanaga
Keyword(s):  
Large Scale ◽  
Aureus Strain ◽  
Affinity Column ◽  
Terminal Sequence ◽  
Active Components ◽  
Simplified Method ◽  
Covalently Linked ◽  
Ultracentrifugal Analysis ◽  
Sepharose 4B ◽  
Single Symmetrical Peak

Staphylocoagulase is known to coagulate human plasma, but not bovine’s, by forming an active molecular complex with prothrombin. However, there is no enough knowledges in respect to the molecular mechanism of prothrombin activation with staphylocoasulase. A new simplified method was developed for the large-scale purification of coagulase from culture filtrates of Staphylococcus aureus. strain st-213, using Sepharose AB covalently linked with bovine prothrombin. This affinity column adsorbed strongly the coagulase, which was eluted with 1.0 M NaSCN. The yield of coagulase activity was in the range of 75 to 85X, The purified coagulase showed a single symmetrical peak by ultracentrifugal analysis (S20,w=6.47), and it gave a single precipitin line against anti-purifled staphylocoagulase serum, as revealed by the immunodiffusion test. However, the preparation was shown to contain three active components by the isoelectric foccusing method, suggesting some microheterogeneity. The molecular weight estimated by SDS-gel electrophoresis was 71,000 (major material). No cystine residue was found in the purified material and its NH2-terminal sequence was Ile-Ile-. It is of interest that staphylocoagulase interacts strongly with bovine prothrombin-Sepharose AB, whereas it does not form any active complex with the prothrombin, unlike human prothrombin.

Sign in / Sign up

Export Citation Format

Share Document