Production and characterization of a new specific antiserum against the taurine putative biosynthetic enzyme cysteine sulfinate decarboxylase.
We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were immunostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.
Almarghini
K
K
INSERM U 171/CNRS URA 1195, Centre Hospitalier Lyon-Sud, Pierre Benite, France.
Barbagli
B
B
Tappaz
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology; D002262:Carboxy-Lyases / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000096:biosynthesis* / Q000276:immunology; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013654:Taurine / Q000096:biosynthesis*
D007106:Immune Sera; D013654:Taurine; D002262:Carboxy-Lyases; C019972:sulfoalanine decarboxylase