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Title: Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

Abstract

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 fractions 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 inhibitmore » 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

Authors:
; ; ;
Publication Date:
Research Org.:
Univ. of Kansas, Lawrence (USA)
OSTI Identifier:
6837957
Alternate Identifier(s):
OSTI ID: 6837957
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Biol. Chem.; (United States); Journal Volume: 263:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GLUTAMIC ACID; MEMBRANE TRANSPORT; PROTEINS; PURIFICATION; TRITIUM COMPOUNDS; BRAIN; CELL MEMBRANES; NERVE CELLS; RECEPTORS; AMINO ACIDS; ANIMAL CELLS; BODY; CARBOXYLIC ACIDS; CELL CONSTITUENTS; CENTRAL NERVOUS SYSTEM; LABELLED COMPOUNDS; MEMBRANE PROTEINS; MEMBRANES; NERVOUS SYSTEM; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANS; SOMATIC CELLS 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Chen, J.W., Cunningham, M.D., Galton, N., and Michaelis, E.K. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. United States: N. p., 1988. Web.
Chen, J.W., Cunningham, M.D., Galton, N., & Michaelis, E.K. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. United States.
Chen, J.W., Cunningham, M.D., Galton, N., and Michaelis, E.K. Tue . "Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes". United States. doi:.
@article{osti_6837957,
title = {Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes},
author = {Chen, J.W. and Cunningham, M.D. and Galton, N. and Michaelis, E.K.},
abstractNote = {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 fractions 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.},
doi = {},
journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 263:1,
place = {United States},
year = {Tue Jan 05 00:00:00 EST 1988},
month = {Tue Jan 05 00:00:00 EST 1988}
}
  • L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. ({sup 14}C)Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of ({sup 14}C)methylamine influx into liposomes reconstitutedmore » with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA{sup +} led to a transient increase in the influx of the lipid-permeable anion probe S{sup 14}CN{sup {minus}}. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the {approximately}69-kDa protein in the function of these ion channels.« less
  • Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-(7-{sup 3}H)indole-3-acetic acid (({sup 3}H)N{sub 3}IAA), the authors identified several IAA-binding proteins with the molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, they were able to selectively extract pm23 from the plasma membrane. They show that auxins and functional analogues compete with ({sup 3}H)N{sub 3}IAA for binding to pm23. They found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1more » as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, they identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. They designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7,000-fold purification.« less
  • Crotoxin and other neurotoxic phospholipase A{sub 2}s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several other to synaptic membranes has been demonstrated previously. In this study the authors used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After {sup 125}I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of {approximately}60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in amore » previous report. The membrane component in the conjugate was shown to be a single-chain protein {approximately}45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation o this adduct with an IC{sub 50} of around 1 {times} 10{sup {minus}8} M. Mojave toxin and some other phospholipase A{sub 2}s were also highly inhibitory to this conjugation, and notexin and others were less effective, while {beta}-bungarotoxin and pancreatic PLA{sub 2} were totally ineffective. They concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A{sub 2}s.« 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
  • 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