Resolution and purification of taurine- and GABA-synthesizing decarboxylases from calf brain.
The present work describes a procedure for the co-purification of cysteine sulfinate decarboxylase (CSAD) and glutamate decarboxylase (GAD) from calf brain. A crude enzyme preparation was first made from brain homogenate by acid precipitation and ammonium sulphate fractionation. Subsequent fractionation of the decarboxylase preparation by cation exchange chromatography on CM-Sepharose CL-6B revealed the existence of a specific CSAD enzyme, which has no GAD activity. The GAD activity peak was found to possess CSAD activity. Further fractionation by gel filtration on Sephacryl S-200 separated the specific CSAD activity into two enzyme forms, one of them having a molecular weight of 150,000 and the other of 71,000. GAD activity was eluted from the gel filtration column in a single peak (mol wt 330,000) and showed CSAD activity. The purification of the specific CSAD enzyme was 920-fold and that of GAD activity 850-fold as compared with the starting material, whole calf brain. SDS gel electrophoresis indicated that the purified CSAD and GAD enzymes consisted of two or more subunits. The crude decarboxylase preparation was analysed by isoelectric focusing in ultra-thin polyacrylamide gel in the pH range 3.5-10.0. The most active fraction of CSAD indicated an isoelectric point of 6.5 and that of GAD 6.8. The pH optimum for CSAD activity in the crude preparation was 7.2 and that for GAD activity 7.9.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D013654:Taurine / Q000096:biosynthesis*; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis*
D009419:Nerve Tissue Proteins; D013654:Taurine; D005680:gamma-Aminobutyric Acid; D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase; C019972:sulfoalanine decarboxylase