scholarly journals Isolation of pathogenesis-related proteins from TMV-infected tobacco and their influence on infectivity of TMV

2010 ◽  
Vol 41 (No. 2) ◽  
pp. 52-57 ◽  
Author(s):  
M. Šindelářová ◽  
L. Šindelář
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Leaf Tissue ◽  
Exchange Chromatography ◽  
Pr Proteins ◽  
Inhibitory Influence ◽  
Pathogenesis Related Proteins ◽  
Molecular Weights ◽  
Pathogenesis Related ◽  
Related Proteins

The composition of pathogenesis-related proteins (PR-proteins) in the intercellular fluid (ICF) and leaf tissue of the hypersensitive tobacco cultivar Xanthi-nc inoculated with Tobacco mosaic virus (TMV), and their inhibitory influence on TMV multiplication were studied. The ICF PR-proteins of infected plants were separated after solubilisation by decreasing gradient of ammonium sulphate, the cell PR-proteins were separated after acidic homogenisation of leaf tissues. The ICF and cell PR-proteins were further purified by ion exchange chromatography on DEAE cellulose. Using discontinuous non-denaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions the PR-proteins were detected. Their molecular weights were estimated by SDS-PAGE. The ICF and cell proteins of infected leaves included PR-proteins of the molecular weights 15–16 kDa (Group 1), 27–28 kDa (Group 3: chitinases) and 36–40 kDa (Group 2a: &#1704-1,3-glucanases). Fractions with different PR-proteins were tested for their effect on infectivity of TMV. Particularly the PR3 and PR2a proteins seem to decrease the infectivity of TMV.

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.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

Light-influenced extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana green tissue induced by various chemicals or prolonged floating on water

1985 ◽  
Vol 63 (7) ◽  
pp. 1276-1283 ◽  
Author(s):  
Alain Asselin ◽  
Jean Grenier ◽  
François Côté
Keyword(s):  
Amino Acids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nicotiana Sylvestris ◽  
Protein Accumulation ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Light Regimes ◽  
Green Tissue ◽  
Pathogenesis Related ◽  
Related Proteins

The extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana tabacum cv. Xanthi-nc and cv. Samsun and in Nicotiana sylvestris was studied by floating leaf disks on solutions (50 μM – 5 mM) of putative inducers at pH 4 to 8 for 72 h. Intercellular fluid b proteins were detected only in green tissue, and optimal conditions required specific light regimes. Amino acids, organic acids, sugars, nucleotides, amines, vitamins, hormones, metals, and several other chemicals were compared with acetylsalicylate at 0.3 mM for the presence of b proteins in intercellular fluid extracts analyzed by native polyacrylamide gel electrophoresis. Several protein and non-protein amino acids and some of their derivatives were inducers in addition to thiamine, thiamine derivatives, and a few other chemicals at concentrations ranging from 200 μM to 5 mM. Various ways of introducing inducers into tissue (floating, injection, infiltration, root uptake) allowed b protein accumulation. Stimulation of b protein accumulation was observed when osmotica (sucrose at 200 mM) were added to inducers. Chelating agents, spermidine, lysine, and hydroquinone could inhibit b protein accumulation induced by L-serine, thiamine, L-α-aminoisobutyrate, and silver nitrate. Results with inducing chemicals and with prolonged floating of tissue on water, which was also conducive to b protein accumulation, support the hypothesis that b protein accumulation is a green tissue generalized response to various forms of prolonged stress.

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.

scholarly journals Induction of Pathogenesis-Related Proteins of Group 1 by Systemic Virus Infections of Nicotiana tabacum L.

1998 ◽  
Vol 18 (2) ◽  
pp. 63-70
Author(s):  
C Röhring
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Pr Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Protein ◽  
Virus Infections ◽  
Pathogenesis Related Proteins ◽  
Pathogenesis Related ◽  
Related Proteins ◽  
Group 1

AbstractThe induction of acid pathogenesis-related proteins (PR-proteins) of group 1 (PR-1) by systemic virus infections of tobacco plants was investigated during a time period between 3 and 19 days after inoculation. Each leaf position was investigated separately. The PR proteins were detected electrophoretically and, in addition, virus protein was detected by Enzyme Linked Immunosorbent Assay (ELISA). Potato virus X (PVX) and potato virus Y (PVY) were found to highly induce PR proteins in N. tabacum L. “Samsun NN”. The same results were obtained when PVX was investigated in N. tabacum L. “Samsun”. The accumulation started later and took place more slowly than during hypersensitive host reaction (HR) of “Samsun NN” to tobacco mosaic virus (TMV). A correlation was found between the accumulation of PR proteins and the accumulation of the virus in the same leaf. Cucumber mosaic virus (CMV) was less effective in PR protein induction than PVX and PVY. Consequently it could be demonstrated that PR proteins do not only appear due to hypersensitive host reactions but also during systemic virus infections.

Identification and characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases

1988 ◽  
Vol 11 (4) ◽  
pp. 529-538 ◽  
Author(s):  
William Nasser ◽  
Marc de Tapia ◽  
Serge Kauffmann ◽  
Shideh Montasser-Kouhsari ◽  
Gérard Burkard
Keyword(s):  
Pr Proteins ◽  
Pathogenesis Related Proteins ◽  
Pathogenesis Related ◽  
Related Proteins ◽  
Identification And Characterization

scholarly journals Isolation, Purification, And Characterization Of Some Cystein Proteases From Bovine Mastites Milk

2009 ◽  
Vol 3 (1) ◽  
pp. 85-97
Author(s):  
K.S. Doosh
Keyword(s):  
Cathepsin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Mastitis ◽  
Deae Cellulose ◽  
Cysteine Proteases ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Molecular Weights

Proteolytic activity of cysteine proteases were studied in bovine mastitis milk, four fractions designated as F1,F2,F3,F4 with cysteine protease activity were separated from leukocytes cell by ion- exchange Chromatography through DEAE-Cellulose The most active fraction F4 was selected for further purification utilizing gel filtration Chromatography on Sephadex G-100 column it has been found that F4 most likely being cathepsin B. purification folds and the enzyme yield was 46.66 and 31.81% respectively . polyacrylamide gel electrophoresis test indicated that the enzyme has been purified to homogeneity by giving a single band . The results of enzyme characterization showed that the molecular weights were 31000 and 30000 Daltons as determined by gel filtration and electrophoresis methods in present of reducing agent SDS- PAGE respectively. The optimum pH for the enzyme activity was 6.0 and it was stable at pH values ranged between 4.5 - 6.5. The enzyme exhibited the maximum activity at 45ºC and the enzyme retained its entire activity over 30 min incubation at 30 -50 C and it retained (50, 20, 10) % of its entire activites over 30 min incubation at (60, 70, 80) C respectively. From this results and results observed from the effect of inhibitor and activator reagents we suggest that enzyme F4 possibly belonged to cathepsin B.

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