Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.
1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569].
D001221:Aspartate Carbamoyltransferase / Q000302:isolation & purification*; D001224:Aspartic Acid; D002852:Chromatography, Ion Exchange; D003080:Cold Temperature; D006863:Hydrogen-Ion Concentration; D012639:Seeds / Q000201:enzymology*; D013696:Temperature; D014908:Triticum / Q000201:enzymology; D014542:Uridine Monophosphate
D001224:Aspartic Acid; D014542:Uridine Monophosphate; D001221:Aspartate Carbamoyltransferase