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Title: Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris

Abstract

The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect {alpha}-amylases but not of plant {alpha}-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, {alpha}-amylase inhibitor ({alpha}Al) 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). All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it. The primary translation product of {alpha}Al is a polypeptide of M{sub r} 28,000. Immunologically cross-reacting glycopolypeptides of M{sub r} 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M{sub r} 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that {alpha}Al is a typical bean lectin-type protein that is synthesized on the rough endoplasmic reticulum, modified in the Golgi, and transportedmore » to the protein storage vacuoles.« less

Authors:
; ;  [1]
  1. (Univ. of California, San Diego, La Jolla (USA))
Publication Date:
OSTI Identifier:
5859528
Resource Type:
Journal Article
Resource Relation:
Journal Name: Plant Physiology; (USA); Journal Volume: 92:3
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AMYLASE; ENZYME INHIBITORS; GLYCOPROTEINS; BIOSYNTHESIS; PHASEOLUS; PHYSIOLOGY; PLANTS; SEEDS; ENZYMES; GLYCOSYL HYDROLASES; HYDROLASES; LEGUMINOSAE; MAGNOLIOPHYTA; MAGNOLIOPSIDA; O-GLYCOSYL HYDROLASES; ORGANIC COMPOUNDS; PROTEINS; SYNTHESIS; 550200* - Biochemistry

Citation Formats

Moreno, J., Altabella, T., and Chrispeels, M.J. Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris. United States: N. p., 1990. Web. doi:10.1104/pp.92.3.703.
Moreno, J., Altabella, T., & Chrispeels, M.J. Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris. United States. doi:10.1104/pp.92.3.703.
Moreno, J., Altabella, T., and Chrispeels, M.J. 1990. "Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris". United States. doi:10.1104/pp.92.3.703.
@article{osti_5859528,
title = {Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris},
author = {Moreno, J. and Altabella, T. and Chrispeels, M.J.},
abstractNote = {The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect {alpha}-amylases but not of plant {alpha}-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, {alpha}-amylase inhibitor ({alpha}Al) 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). All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it. The primary translation product of {alpha}Al is a polypeptide of M{sub r} 28,000. Immunologically cross-reacting glycopolypeptides of M{sub r} 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M{sub r} 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that {alpha}Al is a typical bean lectin-type protein that is synthesized on the rough endoplasmic reticulum, modified in the Golgi, and transported to the protein storage vacuoles.},
doi = {10.1104/pp.92.3.703},
journal = {Plant Physiology; (USA)},
number = ,
volume = 92:3,
place = {United States},
year = 1990,
month = 3
}
  • Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[submore » r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.« less
  • The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has themore » same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.« less
  • Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less
  • The main objectives of this investigation were to study the binding of a lectin from navy beans with the epithelial cells of the rat intestine and to assess the effect of such binding on the ability of the intestine to absorb glucose. A Scatchard plot, based on the binding of /sup 125/I-labeled lectin to isolated intestinal epithelial cells, was used to calculate an association constant (Ka) of 15 x 10(6)M-1 and the number of binding sites per cell, 12 x 10(6). Metabolic studies were conducted over a period of 5 d on groups of rats fed raw or autoclaved navymore » bean flour and casein with or without the purified lectin. Growth, protein digestibility, biological value and net protein utilization were significantly lower in animals that had been fed raw navy bean flour or casein plus lectin than in control groups fed diets containing autoclaved navy bean flour or casein alone. Vascular perfusion was used to measure the rate of uptake of glucose by the intestines of rats that had received the various dietary treatments. The rate of absorption of (/sup 14/C)glucose by intestines from rats fed raw navy bean flour or casein plus lectin was approximately one-half that of their counterparts fed the autoclaved flour or casein alone. These results provide evidence that the lectin, by virtue of its interference with intestinal absorption, is responsible, at least in part, for the nutritional inferiority of raw navy beans.« less
  • Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilizedmore » agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.« less