Subcellular distribution and partial characterization of the three major classes of concanavalin A receptors associated with rat brain synaptic junctions

1980 ◽  
Vol 58 (10) ◽  
pp. 941-951 ◽  
Author(s):  
James W. Gurd
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Heterogeneity ◽  
Molecular Weights ◽  
Receptor Sites ◽  
Synaptic Junctions ◽  
The Individual ◽  
Triton X

Synaptic junctional complexes from rat brain contain three major classes of glycoproteins which react with concanavalin A. They have apparent molecular weights of 110 000 (GP 110) 130 000 (GP 130), and 180 000 (GP 180). They are present in postsynaptic densities but are not found in microsomes, axolemma, synaptic vesicles, or myelin and are present in low concentrations in the Triton X-100 extract obtained during the preparation of synaptic junctions suggesting that they are uniquely localized to the postsynaptic apparatus. Reaction of the individual glycoproteins, partially purified by affinity chromatography on concanavalin A – agarose followed by polyacrylamide gel electrophoresis, showed that GP 130 contained the most receptor sites for concanavalin A per unit of protein followed by GP 180 and GP 110. Of the receptor sites for concanavalin A, 60–70% were subject to hydrolysis by endoglycosidase H indicating that the lectin reacts primarily with polymannose asparagine linked oligosaccharides. Each of the glycoproteins also reacted to varying degrees with the lectins from Lotus tetragonolobus (specific for α-L-fucose), wheat germ (N′-acetyl-D-glucosamine and (or) sialic acid), and lentils (mannose, N′-acetyl-D-glucosamine). Chromatography of 125I-labelled concanavalin A positive glycoproteins on wheat germ Sepharose resolved GP 110 and GP 180 into wheat germ positive and negative components indicating the presence of some structural heterogeneity within these molecular weight classes.

Cross-linking of neuropeptide Y to its receptor on rat brain membranes

1989 ◽  
Vol 256 (3) ◽  
pp. G637-G643 ◽  
Author(s):  
P. J. Mannon ◽  
I. L. Taylor ◽  
L. M. Kaiser ◽  
T. D. Nguyen
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Brain Membranes ◽  
Npy Receptor ◽  
Rat Brain Membranes ◽  
Immature Form ◽  
Triton X

The receptor for neuropeptide Y (NPY) was identified on rat brain membranes after covalent labeling with 125I-NPY using the homobifunctional cross-linkers disuccinimido suberate and disuccinimido dithiobis(propionate) and the heterobifunctional photoactive cross-linker succinimido 4-azidobenzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of two bands at Mr 62,000 and 39,000. Both species showed the same high affinity for 125I-NPY. Exposure to reducing agents did not change the migration of these bands. When the NPY receptor complex was solubilized from the membranes with 1% Triton X-100 and analyzed by gel filtration chromatography, it eluted from a Fractogel TSK 55F column as a peak at approximately 65 kDa. This peak was asymmetric with a shoulder of radioactivity that probably reflects the smaller receptor species. These data indicate that the NPY receptor on rat brain membranes is a monomeric 58-kDa unit (62 kDa minus the mass of the cross-linked NPY) without covalently or noncovalently linked subunits. The smaller 39-kDa species may be an immature form of the 62-kDa species, a second distinct receptor, or a degradation product of the 62-kDa band.

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity

1990 ◽  
Vol 36 (1) ◽  
pp. 33-41 ◽  
Author(s):  
James E. Piechura ◽  
Viswanth P. Kurup ◽  
Laureen J. Daft
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73 000 and 43 000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin–avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that the two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases. Key words: Aspergillus, membrane, allergens, proteases, aspergillosis.

Effect of Sulfur Supply on the Seed Globulin Composition of Various Species of Lupin

1978 ◽  
Vol 5 (5) ◽  
pp. 641 ◽  
Author(s):  
JM Gillespie ◽  
RJ Blagrove ◽  
PJ Randall
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Large Change ◽  
Subunit Composition ◽  
Sulfur Deficiency ◽  
Protein Subunit ◽  
Disulfide Bonding ◽  
Molecular Weights ◽  
Sulfur Supply ◽  
The Individual

The protein level in seeds of six species of lupin, grown either under sulfur deficiency or with adequate sulfur fertilization, is marginally affected by sulfur supply. However, the ratio of total sulfur to total nitrogen in the seed is greatly decreased under sulfur deficiency. This large change in sulfur-to-nitrogen ratio is accompanied by suppression of the synthesis of conglutins α and γ, which contain a significant amount of cyst(e)ine and methionine. The level of protein is maintained by increased synthesis of conglutin β, which normally contains no methionine and a low proportion of cyst(e)ine. These changes in the proportions of the proteins are reflected in the amino acid analyses for the globulin extracts. The changes in protein subunit composition which accompany the differences in the proportions of the proteins have been studied using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results emphasize the differences in subunit composition between lupin species in terms of the number of components, their molecular weights and the importance of disulfide bonding. Two-dimensional electrophoresis, using cellulose acetate and SDS-polyacrylamide gradient gels, has been used to compare the subunit composition of the individual globulins for Lupinus angustifolius and L. elegans at both sulfur levels.

scholarly journals Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

1979 ◽  
Vol 183 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Richard H. Quarles ◽  
Laurence J. McIntyre ◽  
Carol F. Pasnak
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Preceding Paper ◽  
The Other ◽  
Double Labelling ◽  
Myelin Associated Glycoprotein

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [3H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A–Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A–Sepharose and these glycoproteins could be eluted with solutions containing methyl α-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl α-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [3H]fucose-labelled glycoproteins from adult myelin and [14C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205–212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.

scholarly journals Purification, characterization and cellular localization of 5′-nucleotidase from Torpedo electric organ

1987 ◽  
Vol 245 (3) ◽  
pp. 805-810 ◽  
Author(s):  
E J M Grondal ◽  
H Zimmermann
Keyword(s):  
Enzyme Activity ◽  
Concanavalin A ◽  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electric Organ ◽  
Immunohistochemical Analysis ◽  
Maximal Velocity ◽  
Nitrophenyl Phosphate ◽  
Torpedo Electric Organ ◽  
Triton X

5′-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5′-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.

scholarly journals Ultrastructural localization of concanavalin A and wheat germ agglutinin binding sites in adult and embryonic mouse myocardium.

1982 ◽  
Vol 30 (3) ◽  
pp. 193-200 ◽  
Author(s):  
D Gros ◽  
B Bruce ◽  
C E Challice ◽  
J Schrevel
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ultrastructural Localization ◽  
Embryonic Cells ◽  
Con A ◽  
Embryonic Mouse ◽  
Transverse Tubular System ◽  
Receptor Sites ◽  
Ultrathin Sections

The lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), have been used to localize with precision glycosyl residues in adult and embryonic mouse myocardium. They were detected by means of an affinity method using peroxidase and chitobiosylperoxidase, respectively, which then were revealed with 3,3'-diaminobenzidine and H2O2. Exhaustive controls have shown that the binding of Con A and WGA is reversible when experiments are performed with adult specimens (tissue blocks or ultrathin sections of glycol methacrylate-embedded material) or with isolated embryonic cells. Experiments carried out with tissue blocks from embryonic hearts have shown peroxidase binding. This finding is discussed on the basis of the presence of the endogenous lectin-like components in embryonic hearts. Results show that the surface of adult and embryonic myocardial cells specifically bind both Con A and WGA, thus indicating the presence of glycosyl residues similar to alpha-methyl-D-mannoside and N-acetyl-D-glucosamine. In adult heart the transverse tubular system was also labeled. The absence of Con A and WGA receptor sites in the gap junction regions was demonstrated by means of an electron microscope postembedding staining method.

scholarly journals Genetic polymorphism of platelet glycoprotein Ib

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 622-629 ◽  
Author(s):  
M Moroi ◽  
SM Jung ◽  
N Yoshida
Keyword(s):  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Gene Frequencies ◽  
Specific Staining ◽  
Molecular Weights ◽  
Different Types ◽  
Molecular Weight Difference

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos

1979 ◽  
Vol 57 (9) ◽  
pp. 1170-1175 ◽  
Author(s):  
A. C. Cuming ◽  
T. D. Kennedy ◽  
B. G. Lane
Keyword(s):  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Wheat Embryo ◽  
Molecular Weights ◽  
Wheat Embryos ◽  
The Subject ◽  
Mass Isolation ◽  
Gradient Fractionation ◽  
Rabbit Reticulocytes

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS – polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Binding of concanavalin A and ricin to synaptic junctions of rat brain

Nature ◽  
1974 ◽  
Vol 249 (5455) ◽  
pp. 370-371 ◽  
Author(s):  
HELMUT BITTIGER ◽  
HANS PETER SCHNEBLI
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Synaptic Junctions
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