Purification of the subunits of transcarboxylase by affinity chromatography on avidin-sepharose.
Transcarboxylase consists of a central 12 SH subunits each of which is linked to the central subunit by two similar to 1.3 SE biotin carboxyl carrier proteins. The subunits from dissociated transcarboxylase have been difficult to isolate because conditions which stabilize them also promote their reassociation to the intact enzyme. In this paper, we describe the use of avidin-Sepharose to adsorb the enzyme from crude extracts or partially purified transcarboxylase of propionibacteria. After removing impurities by washing the column with phosphate buffer at pH 6.5, in which the transcarboxylase is stable, the enzyme is dissociated first by elution at pH 8 yielding a fraction containing mostly 12 SH subunit which can be rapidly stabilized against dissociation to 6 SH without the problem of reconstitution because the 1.3 SE and most of the 5 SE subunits are not eluted. The second elution is at pH 9 which yields the 5 SE subunit by dissociation from the 1.3 SE biotin subunit and the 1.3 SE subunit remains bound to the avidin. The 12 SH and 5 SE subunits are further purified by glycerol density gradient centrifugation or by chromatography on Bio-Gel. Very active enzyme can be reconstituted from these subunits upon the addition of the 1.3 SE subunit.
D001360:Avidin; D001665:Binding Sites; D001710:Biotin; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008314:Malonates; D011424:Propionibacterium / Q000201:enzymology; D011485:Protein Binding; D011773:Pyruvates; D012685:Sepharose; D014166:Transferases / Q000302:isolation & purification; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D008314:Malonates; D011773:Pyruvates; D001360:Avidin; D001710:Biotin; D012685:Sepharose; D014166:Transferases