Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain

Abstract

The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They include ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter.

Ion-coupled neurotransmitter transport

“…Recently, it has been shown that this family also encodes sodium-dependent transporters that do not use dicarboxylic acids as substrates, but rather neutral amino acids (Shafqat et al 1993;Arriza et al 1993). GLT-1, which encodes the glutamate transporter that was purified (Pines et al 1992;Danbolt et al 1990Danbolt et al , 1992, has 573 amino acids and a molecular mass of 64 kDa, in good agreement with the value of 65 kDa of the purified and deglycosylated transporter . Hydropathy plots are relatively straightforward at the aminoterminal side of the protein and the three different groups have predicted six transmembrane ␣-helices at very similar positions (Storck et al 1992;Pines et al 1992;Kanai and Hediger, 1992).…”

“…Using methodology that enables one to reconstitute many samples simultaneously and rapidly, one of each of the subtypes of the GABA (Radian et al 1986) and the Lglutamate (Danbolt et al 1990) transporters have been purified to apparent homogeneity. Both are glycoproteins and both have an apparent molecular mass of 70-80 kDa.…”

“…They are distinct not only because of their different functional properties. Antibodies generated against the GABA transporter (Radian et al 1986) react (as detected by immunoblotting) only with fractions containing GABA transport activity and not with those containing L-glutamate transport activity (Danbolt et al 1990). The opposite is true for antibodies generated against the glutamate transporter .…”

The mGluR2/3 Agonist LY379268 Blocks the Effects of GLT-1 Upregulation on Prepulse Inhibition of the Startle Reflex in Adult Rats

“…and decapitated; the brains were dissected and the frontal cortex, hippocampus, and striatum were removed. Homogenization and crude synaptic plasma membrane preparation were carried out as described previously (Danbolt et al, 1990). Bio-Rad Protein Assay (Bio-Rad Laboratories, GmbH, München, Germany) and a Beckman DU 530 spectrophotometer (Beckman Coulter, Fullerton, CA, USA) were used to determine the total amount of protein in each homogenate (3-4 measurements per homogenate).…”

Human high affinity, Na+‐dependent l‐glutamate/l‐aspartate transporter GLAST‐1 (EAAT‐1) : gene structure and localization to chromosome 5p11‐p12

“…The Na+-dependent, high affinity glutamate transporters known so far show complex expression patterns in neuronal and glial cells as well as in different regions of central nervous system [1,33,34]. Therefore an understanding of the association of neurodegenerative disorders of excitatory neurons with glutamate transporter mutations on the molecular level is pivotally associated with the knowledge of the gene structure and chromosome localization of all glutamate neurotransmitter transporters known so far.…”