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Title: Specific binding of victorin to a 100-kDa protein from oats

Abstract

Susceptibility of oats to victoria blight, caused by the fungus Cochliobolus victoriae, and sensitivity to the host-specific toxin victorin, produced by the fungus, are controlled by the dominant allele at the Vb locus. It has been postulated that the Vb locus encodes a toxin receptor, although direct evidence for such a receptor is not available. Recent studies on structure-activity relationships of the toxin established a methodology for producing {sup 125}I-labeled victorin. Electrophoretic analysis of proteins from isogenic susceptible and resistant oat genotypes following treatment of leaves with radiolabeled victorin showed that victorin binds in a covalent and a genotype-specific manner to a 100-kDa protein only in susceptible oat leaf slices. This in vivo binding was competitively displaced by reduced victorin, a nontoxic protective compound, and appeared to be correlated with biological activity. In vitro binding to the 100-kDa protein in leaf extracts showed several differences from in vivo binding. Binding was not genotype specific and required a reducing agent that was not required for in vivo binding. Differential centrifugation showed that the 100-kDa victorin binding protein was not a cytosolic protein but was enriched in a high-speed particulate fraction. The data support the hypothesis that the 100-kDa protein is themore » victorin receptor.« less

Authors:
;  [1]
  1. (Cornell Univ., Ithaca, NY (USA))
Publication Date:
OSTI Identifier:
5264406
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA); Journal Volume: 86:11
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MEMBRANE PROTEINS; CROSS-LINKING; RECEPTORS; MOLECULAR STRUCTURE; TOXINS; AUTORADIOGRAPHY; DOSE-RESPONSE RELATIONSHIPS; ELECTROPHORESIS; FUNGAL DISEASES; GENOTYPE; IODINE 125; LABELLED COMPOUNDS; MEMBRANES; OATS; PROTEINS; ANTIGENS; BETA DECAY RADIOISOTOPES; CEREALS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; DISEASES; ELECTRON CAPTURE RADIOISOTOPES; GRASS; INFECTIOUS DISEASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPES; LILIOPSIDA; MAGNOLIOPHYTA; MATERIALS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PLANTS; POLYMERIZATION; RADIOISOTOPES; TOXIC MATERIALS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Wolpert, T.J., and Macko, V.. Specific binding of victorin to a 100-kDa protein from oats. United States: N. p., 1989. Web. doi:10.1073/pnas.86.11.4092.
Wolpert, T.J., & Macko, V.. Specific binding of victorin to a 100-kDa protein from oats. United States. doi:10.1073/pnas.86.11.4092.
Wolpert, T.J., and Macko, V.. Thu . "Specific binding of victorin to a 100-kDa protein from oats". United States. doi:10.1073/pnas.86.11.4092.
@article{osti_5264406,
title = {Specific binding of victorin to a 100-kDa protein from oats},
author = {Wolpert, T.J. and Macko, V.},
abstractNote = {Susceptibility of oats to victoria blight, caused by the fungus Cochliobolus victoriae, and sensitivity to the host-specific toxin victorin, produced by the fungus, are controlled by the dominant allele at the Vb locus. It has been postulated that the Vb locus encodes a toxin receptor, although direct evidence for such a receptor is not available. Recent studies on structure-activity relationships of the toxin established a methodology for producing {sup 125}I-labeled victorin. Electrophoretic analysis of proteins from isogenic susceptible and resistant oat genotypes following treatment of leaves with radiolabeled victorin showed that victorin binds in a covalent and a genotype-specific manner to a 100-kDa protein only in susceptible oat leaf slices. This in vivo binding was competitively displaced by reduced victorin, a nontoxic protective compound, and appeared to be correlated with biological activity. In vitro binding to the 100-kDa protein in leaf extracts showed several differences from in vivo binding. Binding was not genotype specific and required a reducing agent that was not required for in vivo binding. Differential centrifugation showed that the 100-kDa victorin binding protein was not a cytosolic protein but was enriched in a high-speed particulate fraction. The data support the hypothesis that the 100-kDa protein is the victorin receptor.},
doi = {10.1073/pnas.86.11.4092},
journal = {Proceedings of the National Academy of Sciences of the United States of America; (USA)},
number = ,
volume = 86:11,
place = {United States},
year = {Thu Jun 01 00:00:00 EDT 1989},
month = {Thu Jun 01 00:00:00 EDT 1989}
}
  • The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active {sup 125}I derivative of the toxin, the authors identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes wasmore » identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, they developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical.« less
  • The 87-kDa protein, a major specific substrate for protein kinase C, has been purified 500-fold to apparent homogeneity from bovine forebrain supernatant. The purification procedure included batch adsorption to DE-52 (DEAE-cellulose), (NH/sub 4/)/sub 2/SO/sub 4/ precipitation, and chromatography on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Sephacryl S-400, and fast protein liquid chromatography ProRPC. The amino acid composition was notable for its high proportion of alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline (11.3 mol%). The partial specific volume was 0.702 ml/g; the Stokes radius and sedimentation coefficient were 85 A and 2.11 S, respectively. Althoughmore » the relative molecular mass of the protein on NaDodSO/sub 4//8% PAGE was 87-90 kDa, the molecular mass as determined from the above values was 68 kDa. The frictional ratio was 3.2, and the axial ratio was 60, indicating that the 87-kDa protein is an extremely elongated monomer. The purified 87-kDa protein was phosphorylated by purified protein kinase C to a stoichiometry of 2.2 mol of /sup 32/P per mol of 87-kDa protein (calculated using a value of 68 kDa for the molecular mass). Phosphorylation was exclusively on serine residues.« less
  • Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approx. 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractionsmore » were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 /sup +/M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L(/sup 3/H)glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.« less
  • In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, andmore » the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.« less