Pyruvate decarboxylase from Pisum sativum. Properties, nucleotide and amino acid sequences.
To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.
Mücke
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Institut für Biochemie, FB Biochemie/Biotechologie, Martin-Luther-Universität Halle-Wittenberg, Germany.
Wohlfarth
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Fiedler
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Bäumlein
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Rücknagel
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König
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D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019105:Random Amplified Polymorphic DNA Technique; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D011767:Pyruvate Decarboxylase