Reconstitution of petroleum ether soluble wheat lipopurothionin by binding of digalactosyl diglyceride to the chloroform-soluble form

Hernández-Lucas, C.; Caleya, R. Fernandez De; Carbonero, Pilar; Garcı́a-Olmedo, Francisco

doi:10.1021/jf60214a023

Cited by 14 publications

(15 citation statements)

References 10 publications

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“…The characterization of the other lipid components required for solubility in petrol was not pursued further. In all these qualitative solubility experiments, LBPs showed the same properties as those previously reported for the thionins [9]. As judged from the preparative electrophoresis elution profiles (see Fig.…”

Section: Solubility Properties and Purificationsupporting

confidence: 80%

“…Experiments similar to those previously reported for wheat thionins [9] were performed in the case of the LBPs. A preliminary investigation of its solubility in organic solvents was carried out.…”

Section: Solubility Properties and Purificationmentioning

confidence: 95%

“…The proteins reported here (LBPs) were purified from these crude fractions by prep. electrophoresis on 10%polyacrylamidecolumns (1.5 x 10cm)in 0.1 M HOAc buffer, pH 2.9, as described for thionin purification [18].…”

Section: Methodsmentioning

confidence: 99%

“…CHC1 3 and drying in vacuo. DGDG was prepared as described [9]; the amount obtained from 2 g flour was used to reconstitute the amount of Me 2 CO ppt. obtained from 1 g flour.…”

Section: Methodsmentioning

confidence: 99%

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Lipid binding proteins from the endosperms of wheat and Oats

et al. 1984

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Abstract-A protein, designated lipid binding protein (LBP), has been purified from the petrol extracts of wheat and oat endosperms by hydrochloric acid precipitation in a non-polar médium and preparative electrophoresis. The purified LBP appeared to be homogeneous both by electrophoresis in sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gels (MW ca 14 500) and by electrophoresis (PAGE) at pH 3.2. The amino acid composition indicates a high degree of homology between the LBPs from the two sources, as judged by the indexes of Cornish-Bowden and of Harris and Teller. As in the case of thionin, a previously characterized polypeptide from the ether extract, LBP becomes etherinsoluble, chloroform-soluble by precipitation with acetone, and solubility in ether is restored by binding of digalactosyl diglyceride to the chloroform-soluble form.

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Section: Solubility Properties and Purificationsupporting

confidence: 80%

Section: Solubility Properties and Purificationmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

“…CHC1 3 and drying in vacuo. DGDG was prepared as described [9]; the amount obtained from 2 g flour was used to reconstitute the amount of Me 2 CO ppt. obtained from 1 g flour.…”

Section: Methodsmentioning

confidence: 99%

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Lipid binding proteins from the endosperms of wheat and Oats

et al. 1984

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“…The toxicity of thionins to different kinds of organisms and to cells in culture has been investigated over several decades, following the initial reports of their antibiotic properties [4,5]. Toxicity to bacteria [6], yeast [4,7]0 fungi [2,8], animal cells [9,10] and whole animals [10][11][12] has been demonstrated. Current interest in these proteins relates to their possible involvement in plant defense [6].…”

Section: Introductionmentioning

confidence: 99%

Effects of thionins on β‐glucuronidase in vitro and in plant protoplasts

Dı́az

Carmona²,

Garcı́a-Olmedo³

1992

FEBS Letters

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Thionins cause the irreversible inactivation of β‐glucuronidase (GUS) in vitro in a dose‐ and time‐dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally‐added and by transiently‐expressed thionin, suggesting that it interferes with GUS secretion.

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A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis

et al. 1991

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A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. a-purothionin, Urtica dioica agglutinin, and tobacco chitinase.In the purification of biologically active proteins by chromatographic procedures, it is often difficult to assign -with certainty-specific activity to a purified protein. Application of an alternative high resolution separation technique such as polyacrylamide gel electrophoresis (PAGE) can help to confirm comigration of a protein and its activity. For this purpose, different methods have been described for in situ staining of enzymes in gel media [I]. In addition, a technique for detecting antibacterial proteins using duplicate gels was reported [2]. For detection of antifungal proteins, however, no method adapted to PAGE is currently available. Instead, for thin-layer chromatography of low molecular weight antibiotics, in situ detection of antifungal activity is commonly used [3]. Recently, Grenier and Asselin [4] described a technique for detecting proteins with spore lysing activity, whereby fungal spores were used as a "nonliving"substrate. The present communication describes a convenient technique that allows detection of antifungal activity by using growing fungal mycelium as an indicator. In addition, only a single gel is necessary for both activity detection and staining of proteins. The method is illustrated for three antifungal proteins, i.e. a-purothionin (a-PT) from wheat endosperm [5], Urtica dioica agglutinin (UDA) from stinging nettle rhizomes [6], and a basic chitinase (CHI) from tobacco leaves [7].All inorganic salts and organic solutions were of analytical grade. The electrophoresis chemicals were from USB (Cleveland, OH). Pyronin Y and tetracycline were obtained from Sigma (St. Louis, MO). Cefotaxime was from Hoechst (Frankfurt am Main, Germany). Potato dextrose broth (PDB) was purchased from Difco (Detroit, MI). Urtica dioica agglutinin was isolated from stinging nettle rhizomes as previously described [6]. a-Purothionin was purified from wheat flour by the method of Redman and Fisher [8]. Chitinase, isolated from tobacco leaves according to Broekaert et al. [7], actually represents a mixture of two basic isoforms. Ovalbumin was obtained from Sigma. Fungal spores were isolated and stored as described previously [9]. All fungi were grown on six-cereal agar [9] under white fluorescent light.Electrophoresis was carried out in a Midget apparatus (Pharmacia-LKB, Uppsala, Sweden Proteins in the gel were indirectly stained by diffusion blotting. After electrophoresis the gel was overlayed for 10 min with a nitrocellulose paper (0.45 pm, Phar...

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Reconstitution of petroleum ether soluble wheat lipopurothionin by binding of digalactosyl diglyceride to the chloroform-soluble form

Cited by 14 publications

References 10 publications

Lipid binding proteins from the endosperms of wheat and Oats

Lipid binding proteins from the endosperms of wheat and Oats

Effects of thionins on β‐glucuronidase in vitro and in plant protoplasts

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis

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