A non-aqueous method for the sub-cellular fractionation of cotyledons from dormant seeds of Phaseolus vulgaris L.

Begbie, R.

doi:10.1007/bf00389508

Cited by 19 publications

(20 citation statements)

References 19 publications

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“…Immunocytochemical (24) and immunofluorescence (8) studies of Phaseolus vulgaris cotyledons at the light microscopy level showed the presence oflectin in the cytoplasm ofthese cells. Subcellular fractionation studies of this tissue from both dormant and imbibed seeds showed the association of both P. vulgaris seed lectins with the protein bodies (2,3,27). This localization was confirmed by subsequent studies at both the light and electron microscopic levels (22).…”

Section: Discussionsupporting

confidence: 60%

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Subcellular Localizations of Two Dolichos biflorus Lectins

Etzler¹,

Macmillan²,

Scates³

et al. 1984

Plant Physiol.

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The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRMI) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCI, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied IlIIICRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.The seeds of the Dolichos biflorus plant contain a lectin that is specific for terminal nonreducing a-N-acetylgalactosamine residues (13). This lectin is localized in the cotyledons where it accounts for about 10% of the nitrogen in the soluble mature seed extract. Upon germination of the seeds, the seed lectin disappears at about the same rate as the other cotyledon proteins (31). Peptide mapping studies and COOH-terminal amino acid analyses indicate that these subunits are identical to one another, except at their COOH-terminal ends where subunit I appears to be slightly longer than subunit 11 (6,7,29). Despite these similarities, the carbohydrate-binding activity of the lectin appears to reside in subunit 1 (12).Although the seed lectin has not been detected in other parts of the plant at any stage of its life cycle, the stems and leaves of the plant contain a glycoprotein that cross-reacts with antibodies to the seed lectin (31). This CRM' resembles the seed lectin in its amino acid and carbohydrate composition and is a 70,000 mol wt dimer consisting of one subunit with an electrophoretic mobility identical to subunit I of the seed lectin and anot...

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Section: Discussionsupporting

confidence: 60%

“…In an attempt to stabilize the protein bodies during subcellular fractionation, we utilized a modification of a nonaqueous method developed by Begbie (2). Dry D. biflorus seeds were ground for 1 min in a Waring Blendor and 2 g of this coarse powder were transferred to a mortar and pestle and ground to a fine powder.…”

Section: Methodsmentioning

confidence: 99%

Subcellular Localizations of Two Dolichos biflorus Lectins

Etzler¹,

Macmillan²,

Scates³

et al. 1984

Plant Physiol.

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“…Protein extracts (50 ^g) isolated from transgenic tobacco seeds were separated with 12.5% SDS-PAGE, and patatin was visualized using anti-patatin, protein-specific antibodies. Protein bodies were isolated from transgenic tobacco [P-Pat, lanes 1 to 4; P-Pat/P-APat (transgenic plant containing both the cytosolic and vacuolar form), lanes 5 to 8] seeds on a glycerol/ potassium iodide gradient according to Begbie (1979). The protein body-containing fraction was determined by measuring the «-mannosidase activity.…”

Section: Discussionmentioning

confidence: 99%

Expression of mutant patatin protein in transgenic tobacco plants: role of glycans and intracellular location.

1990

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The influence of N-glycosylation and subcellular compartmentation on various characteristics of a vacuolar glycoprotein is described. One member of the patatin gene family was investigated as a model system. Different glycosylation mutants obtained by destroying the consensus site Asn-X-Ser/Thr by oligonucleotide-directed mutagenesis were expressed in leaves of transgenic tobacco plants under the control of a light-inducible promoter. The various patatin glycomutants retained their properties in comparison with the wild-type protein with respect to protein stability, subcellular compartmentation, enzymatic activity, and various physicochemical properties studied showing the N-glycosylation not to be essential for any of these characteristics. To test the importance of the cotranslational transport and the subcellular (vacuolar) location for the properties of the patatin protein, another mutant was constructed in which the signal peptide was deleted, leading to its synthesis and accumulation in the cytosol. Biochemical analysis of this protein in comparison with its vacuolar form again revealed no significant differences with respect to its enzymatic activity or its stability in normal vegetative cells. During seed development, however, the cytoplasmic form was more stable than the vacuolar form, indicating the appearance of proteases specific for the protein bodies of developing seeds.

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“…Bean cotyledons were homogenized in 0.1 M Tris-HCl, 1 mM EDTA with 12% (w/w) sucrose (pH 7.8) (1:10, fresh weight to volume ratio). The microsomal fraction was obtained by chromatography on Sepharose 4B as previously described (28), loaded on top of a 16 to 54% (w/w) linear sucrose gradient in 0.1 M Tris-HCl, 1 mM EDTA (6), and centrifuged at 1 50,000g for 2 h. The gradient was fractionated and an aliquot of each fraction subjected to SDS-PAGE and immunoblotting.…”

Section: Purification Of the Microsomal Fraction And Sucrose Gradientmentioning

confidence: 99%

Characterization of α-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris

Moreno¹,

Altabella²,

Chrispeels³

1990

Plant Physiol.

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The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect a-amylases, but not of plant a-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, a-amylase inhibitor (aAI) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies).

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A non-aqueous method for the sub-cellular fractionation of cotyledons from dormant seeds of Phaseolus vulgaris L.

Cited by 19 publications

References 19 publications

Subcellular Localizations of Two Dolichos biflorus Lectins

Subcellular Localizations of Two Dolichos biflorus Lectins

Expression of mutant patatin protein in transgenic tobacco plants: role of glycans and intracellular location.

Characterization of α-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris

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