Isolation and Characterization of Glucosamine-containing Storage Glycoproteins from the Cotyledons of <i>Phaseolus aureus</i>

Ericson, Mary C.; Chrispeels, Maarten J.

doi:10.1104/pp.52.2.98

Cited by 94 publications

(37 citation statements)

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“…Seeds of mung beans (Phaseolus aureus, Roxb.) were obtained from a local dealer and grown as described in our earlier work (4). The cotyledons were homogenized with a mortar and pestle in 25 mm citrate-phosphate (pH 5.7) containing 10 mm 3-mercaptoethanol and 0.02% sodium azide.…”

Section: Methodsmentioning

confidence: 99%

“…As part of our study on the control mechanism involved in storage protein metabolism in germinating legume seeds (3,4,6), we have analyzed extracts of cotyledons for the presence of inhibitors of the endopeptidase activity. The evidence presented here indicates that the cotyledons contain such inhibitors but suggests that these inhibitors are not involved in the regulation of the metabolism of storage proteins.…”

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Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Baumgärtner

Chrispeels²

1976

Plant Physiol.

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Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.The inhibitory activity against the mung bean endopeptidase is due to the presence of two inhibitors which can be separated by chromatography on Sephadex G-100. The two inhibitors have approximate molecular weights of 12,000 and smaller than 2,000 daltons. The large inhibitor coelutes with trypsin inhibitor on Sephadex G-100, but these two inhibitory activities can be separated by means of a trypsin affinity column.The inhibitory activity disappears slowly from crude extracts incubated at 6 C and more rapidly when the extracts are incubated at 25 C or 37 C. The disappearance of inhibitory activity is accompanied by a rise of the endopeptidase activity, but an examination of the kinetics of these two phenomena suggests that they are not causally related. Fractionation of the ceDular organelles on sucrose gradients shows that the inhibitory activity is not associated with the protein bodies, but rather with the cytosol. Our results suggest that the endopeptidase inhibitor(s) does not regulate the increase in endopeptidase activity which accompanies germination or the metabolism of storage protein. We, therefore, postulate that the inhibitor(s) may function in protecting the cytoplasm from accidental rupturing of the protease-containing protein bodies. geneity and found that more than 95% of the endopeptidase activity in the cotyledons of 5-day-old plants can be accounted for by a single enzyme with an apparent mol wt of 23,000 (Baumgartner and Chrispeels, in preparation).Cotyledons of leguminous seeds are a rich source of protease inhibitors (10). The inhibitors which inhibit proteases of animal or microbial origin (e.g. trypsin inhibitors and chymotrypsin inhibitors) have been studied rather extensively, while relatively little is known about the inhibitors which inhibit endogenous plant proteases (18). Germination is accompanied by a gradual decline in the level of trypsin inhibitors in the cotyledons (7, 16). There is no evidence, however, that the increase in proteolytic activity which also accompanies germination is causally related to the decrease in trypsin-inhibitor activity, and the physiological function of these inhibitors remains unknown (18). Royer et al. (17) have shown recently that the removal of trypsin inhibitors from extracts of cowpea cotyledons caused an increase in the caseolytic activity of these extracts. This led them to suggest that protease inhibitors play a role in the activation of pre-existing proteases and in the control of reserve protein metabolism. A similar mechanism has been proposed for the control of re...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Baumgärtner

Chrispeels²

1976

Plant Physiol.

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“…7S globulin has a molecular weight of 28 and 16 kDa, while 11S globulin has a molecular weight of 40 and 24 kDa. Ericson and Chrispeels [38] reported the subunit molecular weights obtained for 8S and 11S mungbean globulins were 56, 44, and 165 kDa for 11S and 63, 50, 34, and 24 kDa for 8S.…”

Section: Sds-page Profilementioning

confidence: 99%

Structural and functional properties of Maillard reaction products of protein isolate (mung bean,Vigna radiate(L.)) with dextran

Zhou

Zhang

et al. 2017

International Journal of Food Properties

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The objective of this study was to determine the functional and structural properties of Maillard reaction products of mung bean [Vigna radiate (L.)] protein isolate with dextran obtained under the temperature of 80°C or 90°C with different times (0, 1, 2, 3, 4, 5, and 6 h). The mung bean protein isolate-dextran conjugate had better solubility, emulsifying activity, and emulsifying stability than mung bean protein isolate; however, the protein aggregation decreased the solubility, surface hydrophobicity, emulsifying activity, and emulsifying stability of mung bean protein isolate-dextran conjugate. In general, these functional properties of mung bean protein isolate-dextran conjugate obtained at 80°C with lower degree of glycosylation and browning degree were better than mung bean protein isolatedextran conjugate obtained at 90°C. Both vicilin (8S) and legumin type (11S) globulins both participated in the graft reaction, the subunit with a molecular weight of 21 kDa that contributed to 11S globulin might be the most vulnerable to dextran followed by the 60, 32, and 26 kDa subunits. Maillard reaction between mung bean protein isolate and dextran led to a decreased fluorescence intensity and a bathochromic shift. The fluorescence intensity of mung bean protein isolate-dextran conjugate generally decreased, and λ max of mung bean protein isolate-dextran conjugate first increased and then decreased. The grafted mung bean protein isolates had lower α-helix content but higher β-sheet content. As the Maillard reaction between mung bean protein isolate and dextran proceeded, the α-helix contents of the mung bean protein isolate-dextran conjugates were significantly increased and then decreased, and the β-sheet content generally decreased and then increased. Excessive grafted dextran and more protein aggregation was observed in mung bean protein isolate-dextran conjugate obtained at 90°C, which induced structural changes that might have impaired the functions. Our findings suggest that conjugation of mung bean protein isolate with dextran through the Maillard reaction is an effective way of improving the functional properties of mung bean protein isolate for its application in the food industry. ARTICLE HISTORY

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“…They contain the reserve proteins vicilin and legumin (8) as well as a lectin (13) and a variety of acid hydrolases including a-mannosidase, N-acetyl-,/-glucosaminidase, phosphodiesterase, and phospholipase D (21). Germination and seedling growth are accompanied by the hydrolysis of the reserve proteins within the protein bodies.…”

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The Endoplasmic Reticulum of Mung Bean Cotyledons

Wilden¹,

Gilkes²,

Chrispeels³

1980

Plant Physiol.

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The subcelular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organeUle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.The protein bodies of mung bean cotyledons are large (I to 10 ytm) spherical organelles consisting of an amorphous protein matrix surrounded by a limiting membrane. They contain the reserve proteins vicilin and legumin (8) as well as a lectin (13) Recent work shows that a second acid hydrolase, ribonuclease, is also synthesized de novo during seedling growth and also accumulates in the protein bodies (15). Although its function in protein bodies may at first be less obvious, we have recently presented evidence indicating that the acid hydrolases found in protein bodies may function in the autophagic digestion of cytoplasmic structures (21). Here, we present evidence that ribonuclease and vicilin-peptidohydrolase are associated with RER-derived vesicles at the time when these enzymes are being synthesized. We suggest that the ER functions in the transport of these enzymes to the protein bodies. MATERIALS AND METHODS Plant Material and Extraction. Mung bean ( Vigna radiata (L.)Wilczek, cv Berken) seeds were purchased from W. Atlee Burpee Co., Riverside, Calif., and planted in moist Vermiculite after a 24-h imbibition. were harvested at appropriate times and homogenized in 2-4 ml of homogenization buffer (50 mM Tris-HCI [pH 7.5] with 1.0 mm EDTA and either 0.1 or 3.0 mM MgCl2) containing 12% (w/w) sucrose. The homogenate was cleared by centrifugation at 5OOg for 5 min.Purification and Fractionation of Organelles. Organelles were separated from soluble protein by chromatography on a Sepharose 4B (Pharmacia) column (15 mm diameter x 20 cm long). The column was loaded with 3 ml homogenate (10-20 cotyledons) and eluted with the homogenization medium. Fractions of 1.5 ml were collected, and the two fractions containing the most light-sca...

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Isolation and Characterization of Glucosamine-containing Storage Glycoproteins from the Cotyledons of Phaseolus aureus

Cited by 94 publications

References 29 publications

Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Structural and functional properties of Maillard reaction products of protein isolate (mung bean,Vigna radiate(L.)) with dextran

The Endoplasmic Reticulum of Mung Bean Cotyledons

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