ISOLATION AND SEQUENCE-ANALYSIS OF CDNAS ENCODING PHOSPHOENOLPYRUVATECARBOXYKINASE FROM THE PCK-TYPE C-4 GRASS UROCHLOA PANICOIDES

A rabbit antiserum was raised against phosphoenolpyruvate carboxykinas e (PCK) purified from Urochloa panicoides, a PCK-type C-4 monocot. The antiserum was used to screen a cDNA expression library constructed fr om U. panicoides leaf poly(A)(+) RNA. Inserts from immunoreactive clon es were used to rescreen the library and obtain three overlapping cDNA s comprising a 2220 bp composite sequence. The single complete open re ading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainl y due to an N-terminal extension of greater than 65 residues, and reve als high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and dival ent cation binding. The anti-PCK antiserum recognises a 69 kDa polypep tide on immunoblots of either purified PCK or U. panicoides leaf extra cts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunorea ctive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides aris e from the breakdown of PCK during isolation. Northern blot analysis i ndicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradu ally during greening, reaching maximum levels after about 84 h.