UDP galactose:ceramide galactosyltransferase and glutamate/aspartate transporter. Copurification, separation and characterization of the two glycoproteins.
The oligodendrocyte-specific UDP-galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte- and myelin-specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton-X-100-solubilized rat brain microsomes by ion exchange, dye ligand and lectin affinity chromatography as a 64-kDa protein homogenous in SDS/PAGE. It copurified with the brain-specific Na(+)-dependent high-affinity L-glutamate/aspartate neurotransmitter transporter (GLAST-1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high-mannose glycoprotein and GLAST-1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70-kDa isoform of GLAST-1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70-kDa isoform differs only in the degree of glycosylation from the 64-kDa GLAST-1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein-chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST-1 were successfully applied to the cloning of CGT and the first high-affinity glutamate neurotransmitter transporter (GLAST-1) in glia of the central nervous system as well.
Schulte
S
S
Institute of Biochemistry, Medical Faculty, University of Cologne, Germany.
Stoffel
W
W
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D051146:N-Acylsphingosine Galactosyltransferase; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D006023:Glycoproteins; D014404:Tubulin; D005700:Galactosyltransferases; D051146:N-Acylsphingosine Galactosyltransferase