Purification and characterization of an astrocyte GABA-carrier inducing protein (GABA-CIP) released from cerebellar granule cells in culture.
A glycoprotein that induces gamma-aminobutyric acid (GABA) carriers in cultured cerebellar astrocytes was isolated and purified from conditioned media from cultured cerebellar granule cells by anion exchange chromatography, affinity chromatography, and gel filtration. Following gel filtration three fractions corresponding to M(r) 30,000, 60,000, and 240,000 exhibited GABA carrier inducing activity. SDS-PAGE of the M(r) 30,000 fraction revealed under non-reducing conditions three bands corresponding to M(r) 30,000, 60,000, and 120,000. Under reducing conditions only the band corresponding to an M(r) of 30,000 was visible. An identical N-terminal amino acid sequence and amino acid composition was found in the M(r) 30,000 and the M(r) 60,000 fraction from the gel filtration. These results suggest that the protein polymerizes into di- and tetramers. Computer base analysis of the N-terminal amino acid sequence revealed no obvious homology with previously reported N-terminal amino acid sequences. Application of the glycoprotein to cerebellar astrocytes led time and dose dependently to an increased GABA uptake. The effect became maximal after 24 h exposure of the cells. Kinetic analysis of the GABA uptake showed that exposure of the astrocytes to the glycoprotein led to an increase in Vmax for GABA uptake without affecting Km, suggesting an increase in the number of GABA carrier molecules. Addition of actinomycin D together with the glycoprotein abolished this effect suggesting that the glycoprotein acts by stimulating de novo synthesis of GABA carriers. Hence, the newly purified protein secreted from neurons is named GABA-carrier inducing protein (GABA-CIP).