A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.
The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.
Wu
T Y
TY
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Liu
C I
CI
Chang
Y C
YC
D000818:Animals; D001386:Azides; D001921:Brain / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004119:Dimethyl Adipimidate; D004120:Dimethyl Suberimidate; D018698:Glutamic Acid / Q000378:metabolism; D046911:Macromolecular Substances; D008956:Models, Chemical; D008970:Molecular Weight; D011869:Radioligand Assay; D018091:Receptors, AMPA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D013569:Synapses / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D018091:Receptors, AMPA; D004119:Dimethyl Adipimidate; D004120:Dimethyl Suberimidate; D018698:Glutamic Acid; C033169:3-((4-azidophenyl)dithio)propionic N-hydroxysuccinimide; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid