Glycoproteins, enzymatic activities, and b proteins in intercellular fluid extracts from hypersensitive Nicotiana species infected with tobacco mosaic virus

1985 ◽  
Vol 63 (5) ◽  
pp. 928-931 ◽  
Author(s):  
Jean-Guy Parent ◽  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electrophoretic Pattern ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Intercellular Fluid ◽  
Nicotiana Tabacum L ◽  
The One

Intercellular fluid b proteins from hypersensitive Nicotiana tabacum L. cv. Xanthi-nc and N. sylvestris Speg. and Comes infected with tobacco mosaic virus were compared by two-dimensional (2-D) polyacrylamide gel electrophoresis. Except for missing bands b2, b6a, b6b, and b7b, the overall 2-D electrophoretic pattern of N. sylvestris intercellular fluid proteins was similar to the one observed with 'Xanthi-nc' tobacco. Intercellular proteins were also studied by chromatography on con-canavalin A. Glycoproteins corresponding to b6a and b7a proteins of N. tabacum and the [Formula: see text] analog of N. sylvestris were identified. These proteins are probably peroxidase isozymes, as peroxidase activities with the same electrophoretic mobility were detected after polyacrylamide gel electrophoresis. No esterase activity was associated with any b protein band in gels. Esterase activities decreased upon virus infection, but accumulation of b proteins and peroxidase activities increased.

scholarly journals Evaluation of SDS-Polyacrylamide gel electrophoresis and immunostrip technique for detection of Tobacco mosaic virus and Cucumber mosaic virus in IRAQ

2011 ◽  
Vol 5 (2) ◽  
pp. 85-94
Author(s):  
Rakib A. Al-Ani ◽  
Mustafa A. Adhab
Keyword(s):  
Tobacco Mosaic Virus ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Positive Reaction ◽  
Total Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Total Proteins ◽  
Winter Squash

his study was carried out to evaluate the efficiency of electrophoresis on SDS- poly acrylamide slap gel and immunostrip techniques for detection of Tobacco mosaic virus (TMV, genus Tobamovirus) and Cucumber mosaic virus (CMV, genus Cucumovirus, family Bromoviridae), compared with symptoms on diagnostic plants for the two viruses. The results obtained showed that the two methods were effective. The analysis of samples of purified CMV, total proteins from infected cucumber plants, and extracts from infected plants with or without chlorophyll, by electrophoresis on 10% polyacrylamide slap gel containing 0.1% SDS showed two bands of 24 and 26 kd in size, and absent in samples of total protein or extracts of healthy plants. These two proteins represent the coat protein (CP) of CMV. In addition, one 18 kd protein band appeared on SDS- polyacrylamide gel profile which represent the CP of TMV, when samples of purified virus, total protein of infected plants, and plant extracts with or without chlorophyll were analyzed. This band was absent in similar samples from healthy plants. The test of immunostrip specific for CMV showed positive reaction with extracts from melon, cucumber, winter squash, and zucchini infected plants. Similarly, a positive reaction with immunostrip specific for TMV appeared with extracts from tobacco, tomato infected with TMV. No reaction was obtained with healthy plants extract. These results were similar to those obtained from indicator plants for the two viruses.

Analysis of extracellular proteins by native polyacrylamide gel electrophoresis with infiltrated leaf tissue: application to pathogenesis-related proteins

1988 ◽  
Vol 66 (6) ◽  
pp. 1227-1229 ◽  
Author(s):  
Jean Grenier ◽  
François Côté ◽  
Alain Asselin
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Tissue ◽  
Polyacrylamide Gel ◽  
Simple Method ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Pathogenesis Related ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Related Proteins

In addition to polyacrylamide gel electrophoretic analysis of intercellular fluid extracts, a simple method of detection of extracellular pathogenesis-related proteins was based on direct native polyacrylamide gel electrophoresis for acidic and basic proteins with leaf tissue infiltrated with 150 mM sucrose. This technique allowed for the detection of the complete set of tobacco pathogenesis-related proteins without having to extract the intercellular fluid by low-speed centrifugation. A major advantage of the technique is the capacity to observe the distribution of extracellular endogenous or exogenous proteins in the tissue directly subjected to electrophoresis.

Detection of 10 additional pathogenesis-related (b) proteins in intercellular fluid extracts from stressed 'Xanthi-nc' tobacco leaf tissue

1987 ◽  
Vol 65 (3) ◽  
pp. 476-481 ◽  
Author(s):  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Gel Electrophoresis ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Tissue ◽  
Polyacrylamide Gel ◽  
Basic Proteins ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Pathogenesis Related ◽  
Related Proteins

By using two-dimensional polyacrylamide gel electrophoresis, 10 additional pathogenesis-related proteins (b6c, b9c, b10a, b10b, b11a, b11b, b12, b13, b14, b15) were found in intercellular fluid extracts of stressed 'Xanthi-nc' tobacco leaf tissue. Proteins were identified as extracellular pathogenesis-related stress proteins by polyacrylamide gel electrophoresis analysis of three successive intercellular fluid extracts compared with homogenates before and after making intercellular fluid extracts. Four proteins (b12, b13, b14, b15) were only resolved by using native polyacrylamide gel electrophoresis for basic proteins in the first dimension gel and they were best extracted in 0.05 M Tris–HCl (pH 7.5) and 0.05 M CaCl2 as the infiltration buffer. The four basic proteins were found in intercellular fluid extracts from leaf tissue subjected to the same types of chemical inducers as previously described for tobacco pathogenesis-related proteins. Their accumulation was inhibited by basic amino acids or spermidine (1 mM) and they were resistant to endogenous and exogenous (trypsin, subtilisin) proteolysis. They did not bind to concanavalin A – Sepharose. These findings indicate that at least 23 proteins accumulate extracellularly after various types of stress in 'Xanthi-nc' tobacco green tissue. These proteins probably represent several groups or families of plant stress proteins.

Effect Of Heparin On The Thrombin-Antithrombin Reaction Product

1981 ◽  
Author(s):  
Isidore Danishefsky ◽  
Michael S Bender
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Lower Molecular Weight ◽  
Primary Complex ◽  
Synthetic Substrate ◽  
The One

The characteristics of the primary complex (C-l) formed between thrombin and antithrombin in the absence and presence of heparin, were investigated. Each of the complexes were isolated by gel-filtration of the reaction mixture on Sephadex G-100.Analyses by SDS-polyacrylamide gel electrophoresis showed that thrombin causes the successive degradation of both complexes to lower molecular weight products C-2 and C-3, respectively. C-l that was formed in the absence of heparin also undergoes spontaneous direct degradation at pH 7.5, to a complex that is similar to C-3. Additionally, this C-l dissociates very slowly to release thrombin, as demonstrated by its action on a synthetic substrate. Treatment of C-l with 1M NH2OH results in its breakdown to thrombin and antithrombin. The complex formed in the presence of heparin differs from the one formed without heparin, in that it does not exhibit any measurable dissociation and does not undergo breakdown to the C-3-type product. Moreover, whereas C-l formed in the absence of heparin is decomposed completely by 1M NH2OH, the complex formed in the presence of heparin undergoes only partial breakdown even with 2M NH2OH. Addition of heparin to C-l originally produced in the absence of heparin, has no effect on its properties.The results thus indicate that heparin influences the mode of binding between thrombin and antithrombin as well as the rate of their interaction.

scholarly journals Glycosidases induced in Aspergillus tamarii. Secreted α-d-galactosidase and β-d-mannanase

1984 ◽  
Vol 219 (3) ◽  
pp. 857-863 ◽  
Author(s):  
A Civas ◽  
R Eberhard ◽  
P Le Dizet ◽  
F Petek
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Preceding Paper ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Aspergillus Tamarii ◽  
Electrophoretic Method ◽  
Single Protein

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.

scholarly journals Co-purification of galactosyltransferases from chick-embryo liver

1985 ◽  
Vol 227 (2) ◽  
pp. 573-582 ◽  
Author(s):  
K Furukawa ◽  
S Roth
Keyword(s):  
Chick Embryo ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Cellulose Acetate Electrophoresis ◽  
Km Value ◽  
Alpha Lactalbumin

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a β-(1→4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a β-(1→3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1→4)-transferase] and the AsOSM enzyme [(1→3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1→4)-transferase, is 4mM; for the (1→3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.

Detection of pathogenesis-related proteins (PR or b) and of other proteins in the intercellular fluid of hypersensitive plants infected with tobacco mosaic virus

1984 ◽  
Vol 62 (3) ◽  
pp. 564-569 ◽  
Author(s):  
Jean-Guy Parent ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Chenopodium Quinoa ◽  
Leaf Tissue ◽  
Electrophoretic Analysis ◽  
Intercellular Fluid ◽  
Molecular Weights ◽  
Pathogenesis Related ◽  
The One ◽  
Related Proteins

In tobacco mosaic virus infected hypersensitive Nicotiana species, the pathogenesis-related (PR) or b proteins were found with other proteins in the intercellular fluid of leaf tissue. Analysis of leaves from mock-inoculated plants did not reveal the presence of detectable amounts of proteins in the intercellular fluid. The presence of proteins in the intercellular fluid seems to be widespread since 10 proteins were detected in tobacco mosaic virus infected Chenopodium quinoa Willd. These proteins were found not only in inoculated tissue but also in the intercellular fluid of uninoculated upper leaves. A two-dimensional gel electrophoretic analysis of intercellular fluid proteins from N. tabacum L. cv. Xanthi-nc infected leaves showed that the relative molecular weight of protein b2 is larger (14 700) than the one of b1 and b3 (14 200). Other proteins, ranging in molecular weights from 12 500 to 36 300, could also be detected. We postulate that the presence of rather large amounts of proteins, including the well-characterized b proteins, in the intercellular fluid of infected hypersensitive leaves reflects overall changes in cell-to-cell interactions and in the cell wall metabolism.

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