PubMed · 7516125
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E M Schuman, D V Madison. 1994. Nitric oxide and synaptic function.. https://doi.org/10.1146/annurev.ne.17.030194.001101
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In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.
A novel type of L-amino acid oxidase from Synechococcus PCC6301 was purified and subjected to amino acid sequence analysis. Since the N-terminus of the L-amino acid oxidase protein was not accessible for Edman degradation, the protein was partially hydrolysed and a contiguous sequence of 17 amino acid residues was obtained from an endogenous peptide fragment. Based on the partial peptide sequence two oligonucleotides were designed, which were used as probes in Southern hybridization experiments in order to identify the corresponding aoxA gene. The aoxA gene was isolated from a size-fractionated genomic library of Synechococcus PCC6301 and subsequently sequenced. From the nucleotide sequence (data base accession number Z48565) it can be deduced that the L-amino acid protein consists of 355 amino acid residues resulting in a molar mass of 39.2 kDa. The calculated isoelectric point of the protein is 9.81. The L-amino acid oxidase from Synechococcus PCC6301 shows low homologies to other flavin oxidases/dehydrogenases, especially amine oxidases, but no homologies to other so far sequenced L- or D-amino acid oxidases.
In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.