Isolation, characterization, and purification to homogeneity of a rat brain protein (GABA-modulin).

Abstract

GABA modulin is a brain neuropeptide that appears to modulate specific high-affinity (20 nM) GABA recognition sites in brain. When added to crude synaptic membranes this peptide inhibits binding of [3H]GABA to the high-affinity site and prevents facilitation of [3H]diazepam binding elicited by GABA. GABA-modulin has been purified to homogeneity by ammonium sulfate precipitation, gel chromatography, and reverse-phase HPLC [high performance liquid chromatography]. Homogeneity was confirmed by a variety of means, including chromatography under 4 different HPLC conditions, 2 different polyacrylamide gel electrophoreses and end group analysis. Purified GABA-modulin contains .apprx. 126 amino acids and has a MW of 16,500. The GABA-modulin molecule contains an abundance of hydrophilic basic residues, and neither cysteine nor GABA is present. End group analyses of GABA-modulin showed that histidine is the free COOH terminus and the NH2 terminus is blocked. GABA-modulin specifically blocked both [3H]GABA binding to synaptic membranes (IC50, 0.5 .mu.M) and GABA-stimulated [3H]diazepam binding; the binding of [3H]GABA to low-affinity sites was not affected.