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.

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells. II. In vitro Incorporation of Galactosyl Residues From UDPgalactose Into Polymeric Products

1982 ◽  
Vol 9 (1) ◽  
pp. 31 ◽  
Author(s):  
T Mascara ◽  
GB Fincher
Keyword(s):  
Cell Walls ◽  
Membrane Fraction ◽  
Lolium Multiflorum ◽  
Apparent Molecular Weight ◽  
Arabinogalactan Protein ◽  
Methylation Data ◽  
Myeloma Protein ◽  
Mouse Myeloma ◽  
Galactosyl Residues

When mixed-membrane fractions from suspension-cultured Lolium multiflorum endosperm cells are incubated in vitro with UDP-[14C]galactose, 66% ethanol-insoluble products of apparent molecular weight greater than 60 000 are labelled in both galactosyl and glucosyl residues, suggesting that an active UDPgalactose 4-epimerase is present on the membrane fraction. The epimerase can be inhibited with ADPribose, to produce polymeric material in which [14C]galactosyl residues pre-dominate. While some of these residues appear to be associated with glycoproteins, affinity chromatography of the products on mouse myeloma protein J539-Sepharose provides evidence that β-galactans containing 1,6-linkages are amongst the products. Monosaccharide analyses and methylation data indicate that the mixed-membrane preparations contain associated polysaccharide of structure analogous to the 1,3;1,4-β-glucans, arabinoxylans and arabino-3,6-galactans normally found in cell walls or secreted into the medium.

scholarly journals Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells

1989 ◽  
Vol 264 (3) ◽  
pp. 857-862 ◽  
Author(s):  
P A Gleeson ◽  
M McNamara ◽  
R E H Wettenhall ◽  
B A Stone ◽  
G B Fincher
Keyword(s):  
Polyclonal Antibodies ◽  
Lolium Multiflorum ◽  
Italian Ryegrass ◽  
Arabinogalactan Protein ◽  
Myeloma Protein ◽  
Protein Core ◽  
Liquid Suspension ◽  
Peptide Component ◽  
Immunodominant Epitopes

An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.

scholarly journals Biosynthesis of arabinogalactan-protein in Lolium multiflorum (Italian ryegrass) endosperm cells. Subcellular distribution of galactosyltransferases

1984 ◽  
Vol 218 (2) ◽  
pp. 633-636 ◽  
Author(s):  
A Schibeci ◽  
A Pnjak ◽  
G B Fincher
Keyword(s):  
Golgi Apparatus ◽  
Subcellular Distribution ◽  
Lolium Multiflorum ◽  
Italian Ryegrass ◽  
Arabinogalactan Protein ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Carbohydrate Component ◽  
Myeloma Protein ◽  
Putative Marker

Intracellular membranes from protoplasts of Italian-ryegrass (Lolium multiflorum) endosperm cells have been fractionated on sucrose density gradients and identified on the basis of putative-marker-enzyme assays. Galactosyltransferases capable of incorporating galactose from UDP galactose into 66% ethanol-soluble products are associated with all membrane fractions. Affinity chromatography of the ethanol-insoluble products on (murine myeloma protein J539)-Sepharose reveals that the enzymes responsible for the synthesis of polymers containing (1→6)-beta-D-galactose residues are associated exclusively with subcellular fractions enriched in Golgi-derived membranes. This suggests that the Golgi apparatus plays an important part in the synthesis of the carbohydrate component of the ryegrass arabinogalactan-protein.

Difference of [Ca2+]i Movements in Platelets Stimulated by Thrombin and TRAP: the Involvement of αIIbβ3-Mediated TXA2 Synthesis

1998 ◽  
Vol 79 (06) ◽  
pp. 1184-1190 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Kayoko Senzaki ◽  
Akito Tanaka ◽  
Mitsuru Okubo ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Fibrinogen Binding ◽  
Adp Release ◽  
The Difference ◽  
Txa2 Receptor Antagonist ◽  
High Level ◽  
Inhibition Studies ◽  
Peak Increase ◽  
Second Peak ◽  
Assay Conditions

SummaryThis study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of αIIbβ3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with αIIbβ3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that αIIbβ3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with αIIbβ3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to αIIbβ3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.

Fine structure of the arabinogalactan-protein from Lolium multiflorum

1987 ◽  
Vol 162 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Antony Bacic ◽  
Shirley C. Churms ◽  
Alistair M. Stephen ◽  
Peter B. Cohen ◽  
Geoffrey B. Fincher
Keyword(s):  
Fine Structure ◽  
Lolium Multiflorum ◽  
Arabinogalactan Protein

scholarly journals Biosynthesis of immunoglobulin A (IgA). Secretion and addition of carbohydrate to monomer and polymer forms of a mouse myeloma protein

1973 ◽  
Vol 136 (3) ◽  
pp. 589-596 ◽  
Author(s):  
E. Della Corte ◽  
R. M. E. Parkhouse
Keyword(s):  
Plasma Cell ◽  
Molecular Species ◽  
Specific Antibody ◽  
Early Stage ◽  
Immunoglobulin A ◽  
Carbohydrate Composition ◽  
Myeloma Protein ◽  
J Chain ◽  
Co Precipitation ◽  
Mouse Myeloma

Cell suspensions of mouse plasma-cell tumour MOPC 315 secreting predominantly IgA (immunoglobulin A) monomer and dimer were incubated with radioactive leucine, mannose, galactose and fucose for various periods of time. The amounts of secreted and intracellular immunoglobulins were measured by co-precipitation with specific antibody, and the molecular species present were assessed by electrophoresis in polyacrylamide gels. Analysis of the secreted myeloma protein demonstrated that monomer and dimer IgA molecules are identical with respect to carbohydrate composition and rate of secretion. Within the cell, the myeloma protein is almost entirely accounted for by monomer units which either leave the cell as such or are polymerized with the addition of J chain close to the time of secretion. The results support the concept of a stepwise addition of carbohydrate residues to IgA immunoglobulin during the process of secretion. Similar patterns of carbohydrate assembly were found for the monomer or dimer molecules. Mannose residues are added at an early stage, whereas fucose is added close to the time of secretion. Galactose is also added early, but some may also be incorporated at a later stage. Control of IgA polymerization is considered unlikely to reflect regulation at the level of carbohydrate addition, and it is suggested that the critical controlling factor is the J chain.

scholarly journals Helper T cell recognition of the variable domains of a mouse myeloma protein (315). Effect of the major histocompatibility complex and domain conformation.

1982 ◽  
Vol 155 (6) ◽  
pp. 1587-1596 ◽  
Author(s):  
T Jørgensen ◽  
K Hannestad
Keyword(s):  
T Cells ◽  
Cell Response ◽  
Helper T Cells ◽  
Helper Cell ◽  
Spleen Cells ◽  
Myeloma Protein ◽  
Helper Cells ◽  
Low Responders ◽  
High Responsiveness ◽  
Mouse Myeloma

We have examined the recognition of the variable (V) domain of the heavy (VH) and light (V lambda 2) chains of mouse myeloma protein 315 by helper T cells. Mice were primed with the isolated V domain in complete Freund's adjuvant, and carrier (V domain)-primed spleen cells were transferred together with hapten (NIP)-primed spleen cells to recipient mice that were boosted with NIP3-Fab-315. The helper cell response to both domains was governed by H-2-linked immune response (Ir) genes, and VH-315 and V lambda 2 displayed different Ir phenotypes. H-2k conferred high responsiveness to VH on three different genetic backgrounds, BALB/c, C3H, and B10; mice of the d and b haplotypes were low responders. Conversely, H-2d conferred high responsiveness to V lambda 2 on two backgrounds, BALB/c and C3H, whereas mice of the k haplotype were low responders to this domain. Non-H-2 genes of the B10 background extinguished the helper cell response to V lambda 2 in animals with the high responder d haplotype. The VH Ir gene mapped to the K-A interval of the H-2 complex. Unfolded (completely reduced and alkylated) V domains primed helper cells as efficiently as folded domains for responses to NIP3-Fab-315, indicating that the helper cells recognized an antigenic determinant that was not conformation-dependent. The data indicate that there exists helper T cells which recognize each member of the M315 pair of V domains independent of the other, and that these V domains are recognized like conventional extrinsic protein antigens.

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 Crystallization of the Fv fragment of mouse myeloma protein M315

1979 ◽  
Vol 181 (2) ◽  
pp. 497-499 ◽  
Author(s):  
R Aschaffenburg ◽  
D C Phillips ◽  
D R Rose ◽  
B J Sutton ◽  
S K Dower ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Unit Cell ◽  
Poly Ethylene Glycol ◽  
Myeloma Protein ◽  
Space Group ◽  
Cell Dimensions ◽  
Poly Ethylene ◽  
Unit Cell Dimensions ◽  
Glycol Solution ◽  
Mouse Myeloma

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.

scholarly journals Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors.

1982 ◽  
Vol 94 (2) ◽  
pp. 406-413 ◽  
Author(s):  
M Simionescu ◽  
N Simionescu ◽  
G E Palade
Keyword(s):  
Binding Sites ◽  
Ricinus Communis ◽  
Capillary Endothelium ◽  
Anionic Sites ◽  
Lectin Receptors ◽  
Fenestrated Endothelium ◽  
Phosphate Buffered Saline ◽  
Soybean Lectin ◽  
Galactosyl Residues

Lectins conjugated with either peroxidase or ferritin were used to detect specific monosaccharide residues on the luminal front of he fenestrated endothelium in the capillaries of murine pancreas and intestinal mucosa. The lectins tested recognize, if accessible, the following residues: alpha-N-acetylgalactosaminyl (soybean lectin), beta-D-galactosyl (peanut agglutinin [PA] and Ricinus communis agglutinin-120 [RCA]), beta-N-acetylglucosaminyl and sialyl residues (wheat germ agglutinin [WGA]), alpha-L-fucosyl (lotus tetragonolobus lectin), and alpha-D-glucosyl and beta-D-mannosyl (concanavalin A [ConA]). Thi labeled lectins were introduced by perfusion in situ after thoroughly flushing with phosphate-buffered saline the microvascular beds under investigation. Specimens were fixed by perfusion, and subsequently processed for peroxidase detection and electron microscopy. Control experiments included perfusion with: (a) unlabeled lectin before lectin conjugate; (b) labeled lectin together with the cognate hapten sugar, and (c) horseradish peroxidase or ferritin alone. Binding sites were found to be relatively homogeneously distributed on the plasmalemma proper, except for Lotus tetragonolobus lectin and Con A, which frequently bound in patches. Plasmalemmal vesicles, transendothelial channels, and their associated diaphragms were particularly rich in residues recognized by RCA and PA (beta-D-galactosyl residues) and by WGA (beta-N-acetylglucosaminyl residues). Receptors for all lectins tested appeared to be absent or considerably less concentrated on fenestral diaphragms. The results reported here extend and complement previous findings on the existence of microdomains generated by the preferential distribution of chemically different anionic sites (Simionescu et al., 1981, J. Cell Biol., 9:605-613 and 614-621).

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