Endogenous polypeptide-chain length and partial sequence of aspartate transcarbamoylase from wheat, characterised by immunochemical and cDNA methods

Abstract

Aspartate transcarbamoylase (ATCase) is purified from wheat germ as a monofunctional trimer of 36 kDa chains. The possibility that this may be a proteolytic fragment of a large endogenous complex in which ATCase is covalently fused to other pyrimidine-pathway enzymes (such as exists in animals or fungi) was tested. Examination of a rabbit antiserum raised against the purified enzyme confirmed the presence of anti-(wheat ATCase) antibodies by several independent methods. Extracts of wheat seedlings prepared under non-proteolysing conditions were challenged by the antiserum, and in some cases by purified anti-(36 kDa ATCase) antibodies, using immunoblotting techniques. The 36 kDa species was the dominant immunopositive polypeptide. However, the extract also contained small amounts of two larger (45 and 55 kDa) immunopositive polypeptides, as well as traces of polypeptides smaller than 36 kDa, which were assumed to be minor proteolytic products. Neither of the 45 or 55 kDa polypeptides is large enough to also incorporate a carbamoyl phosphate synthetase or dihydroorotase of the sizes found in other organisms. They may be targeted precursors of ATCase with intact leader sequences. A screen of a wheat cDNA expression library by the anti-(ATCase) serum yielded a single positive clone which was shown, by DNA sequencing, to be a concatemer of two cDNAs, one of which encoded a partial ATCase. Northern analysis using this clone as probe identified two transcripts of about 1.3 and 1.0 kbp, but showed no evidence of a transcript of 2 kbp or greater which would be required to encode a bifunctional polypeptide. These results confirm that wheat ATCase is not translationally fused to dihydroorotase or carbamoylphosphate synthetase, as it is in animals and fungi. The deduced amino-acid sequence of the partial wheat ATCase is compared with the catalytic chain of the ATCase from Escherichia coli and with other ATCases.