GLYCINE DECARBOXYLASE COMPLEX FROM HIGHER-PLANTS - MOLECULAR-CLONING,TISSUE DISTRIBUTION AND MASS-SPECTROMETRY ANALYSES OF THE T-PROTEIN

cDNA clones encoding the precursor of the T protein of the glycine dec arboxylase complex have been isolated from a pea leaf cDNA library in lambda gt11. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N-termin al cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness o f the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mas s obtained (40966 +/- 5 Da) was the value predicted from the cDNA (409 61 Da). Secondly, the purified T protein was chemically cleaved with c yanogen bromide and the peptide fragments were analysed by high-perfor mance liquid chromatography/electrospray ionization mass spectrometry and/or fast-atom-bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these tec hniques were very close to the values calculated from the predicted pr imary structure. Thirdly, microsequencing of some of these peptides, w hich represent 35% of the total protein, fits perfectly with the prima ry structure deduced from the cDNA. In the present HPLC/electrospray i onization MS studies we never detected the presence of covalently boun d tetrahydropteroylpolyglutamate (H-4PteGlu(n)), either in the native T protein or in the different peptide fragments generated by the chemi cal cleavage. The absence of H-4PteGlu(n) bound to the T protein in ou r experimental conditions demonstrates that H-4PteGlu(n) is not covale ntly linked to the T protein. Northern blot analysis showed that the s teady-state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (almost-equa l-to 8-10-fold higher). Surprisingly, a non-negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA e ncoding the H protein was not detectable. Western blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex wer e present in roots whereas the H protein was not detectable. Southern hybridization to pea genomic DNA indicated the presence of a single ge ne encoding the T protein of the glycine decarboxylase complex in the haploid genome.