COMPARTMENTALIZATION IN THE COTYLEDONARY CELLS OF <i>PHASEOLUS VULGARIS</i> L. SEEDS: A DIFFERENTIAL SEDIMENTATION STUDY

Pusztai, Á.; Croy, R. R. D.; Grant, G.; Watt, William B.

doi:10.1111/j.1469-8137.1977.tb02181.x

Cited by 39 publications

(10 citation statements)

References 34 publications

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“…It is possible that the protein bodies may have been ruptured in the aqueous medium employed for the experiment. Such apparent protein body disruption was found in the above microscopy experiments when the cotyledon sections were treated in aqueous solutions without prior fixation, and changes in protein bodies in aqueous sucrose have been reported by investigators working with other legumes (26,35).…”

Section: Methodsmentioning

confidence: 86%

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: Methodsmentioning

confidence: 86%

Subcellular Localizations of Two Dolichos biflorus Lectins

Etzler¹,

Macmillan²,

Scates³

et al. 1984

Plant Physiol.

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“…This treatment also destroyed most of the trypsin inhibitor activity leading to the erroneous conclusion that the majority of this activity was associated with the protein bodies. Two other studies (8,15) in which protein bodies were isolated by differential centrifugation led to the conclusion that trypsin inhibitory activity is mostly found in the cytoplasm although some of it is associated with protein bodies.…”

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confidence: 99%

Trypsin Inhibitor in Mung Bean Cotyledons

Chrispeels¹,

Baumgärtner²

1978

Plant Physiol.

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Trypsin inhibitor was purHied to homogeneity from seeds of the mung bean ( Vigna radiats ILl Wilczek). The protease inhibitor has the foUowing properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel ifitration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptdase in the cotyledon of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of,B-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts. Seed germination and the subsequent growth of the young seedlings are characterized by the breakdown of the reserve proteins of the seed. This breakdown is accompanied at an enhancement of protease-especially endopeptidase-activity, as well as by a decrease in protease inhibitor activity. Seeds of the Leguminosae, the Graminae, and the Solanaceae are an abundant source of protease inhibitors, and especially trypsin-or chymotrypsin-inhibitors, but the role of these inhibitors in the physiology of the plants is not understood. It has been proposed that these proteinaceous inhibitors may be storage proteins, that they play a role in masking the activity of preexisting plant proteases, and that they protect the seeds against pathogens and phytophagous insects (for reviews see Liener and Kakade (11) Shain and Mayer (20) reported that a trypsin inhibitor in lettuce seeds inhibits an endogenous trypsin-like enzyme which increases during germination, but the function of this enzyme in reserve protein breakdown is unknown. Inhibitors of endogenous plant proteases have been found in rice (9), barley (14), and in mung bean (1). However, several studies led to the conclusion that the great increase in endopeptidase in the storage tissues during seed germination cannot be accounted for by the inactivation of an "equivalent amount" of inhibitors (1, 14).Recently Miege and collaborators (13,17,18)

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“…The fact that the metalloproteinase is present as a complex with its inhibitor is confirmed by the inability of extracts of the protein bodies to autolyze the 13S globulin. It should be noted that the absence or the low level of autolysis of storage proteins in extracts of protein bodies of dry seeds was also observed in legumes, namely mung beans (Harris and Chrispeels 1975) and French beans (Pusztai et al 1977). However, the authors explained this fact by the absence in dry seeds of enzymes capable of hydrolysing storage proteins.…”

Section: Discussionmentioning

confidence: 47%

Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds

Elpidina

Voskoboynikova

Belozersky

et al. 1991

Planta

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Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl4. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein - the 13S globulin - of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.

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COMPARTMENTALIZATION IN THE COTYLEDONARY CELLS OF PHASEOLUS VULGARIS L. SEEDS: A DIFFERENTIAL SEDIMENTATION STUDY

Cited by 39 publications

References 34 publications

Subcellular Localizations of Two Dolichos biflorus Lectins

Subcellular Localizations of Two Dolichos biflorus Lectins

Trypsin Inhibitor in Mung Bean Cotyledons

Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds

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