Solubilisation and characterisation of a putative quisqualate-type glutamate receptor from chick brain.
The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Henley
J M
JM
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, England.
Barnard
E A
EA
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002845:Chromatography; D003902:Detergents / Q000378:metabolism; D007051:Ibotenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012995:Solubility; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
D003902:Detergents; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D007051:Ibotenic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid