Activities | Families | Sequences | Fold types | References | Help
B6db references: 8694755

type Journal Article
authors Niemann, G.; von Besser, H.; Walter, R. D.
title Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein
journal Biochem J
sel selected
ui 8694755
year (1996)
volume 317
number Pt 1
pages 135-40
keywords Amino Acid Sequence
abstract A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha- diffluoromethylornithine, alpha-hydrazino-ornithine and alpha- methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.
last changed 2009/06/08 14:05

B6db references