ENDOGENOUS POLYPEPTIDE-CHAIN LENGTH AND PARTIAL SEQUENCE OF ASPARTATETRANSCARBAMOYLASE FROM WHEAT, CHARACTERIZED BY IMMUNOCHEMICAL AND CDNA METHODS

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 ATCas e is covalently fused to other pyrimidine-pathway enzymes (such as exi sts in animals or fungi) was tested. Examination of a rabbit antiserum raised against the purified enzyme confirmed the presence of anti-(wh eat ATCase) antibodies by several independent methods. Extracts of whe at seedlings prepared under non-proteolysing conditions were challenge d by the antiserum, and in some cases by purified anti-(36 kDa ATCase) antibodies, using immunoblotting techniques. The 36 kDa species was t he dominant immunopositive polypeptide. However, the extract also cont ained small amounts of two larger (45 and 55 kDa) immunopositive polyp eptides, 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 phos phate synthetase or dihydroorotase of the sizes found in other organis ms. They may be targeted precursors of ATCase with intact leader seque nces. A screen of a wheat cDNA expression library by the anti-(ATCase) serum yielded a single positive clone which was shown, by DNA sequenc ing, to be a concatemer of two cDNAs, one of which encoded a partial A TCase. Northern analysis using this clone as probe identified two tran scripts of about 1.3 and 1.0 kbp, but showed no evidence of a transcri pt of 2 kbp or greater which would be required to encode a bifunctiona l polypeptide. These results confirm that wheat ATCase is not translat ionally fused to dihydroorotase or carbamoylphosphate synthetase, as i t is in animals and fungi. The deduced amino-acid sequence of the part ial wheat ATCase is compared with the catalytic chain of the ATCase fr om Escherichia coli and with other ATCases.