Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as...

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Bibliographic Details
Published in:Biochemical journal 1996-09, Vol.318 ( Pt 3) (3), p.879-882
Main Authors: Mitchell, J L, Choe, C Y, Judd, G G
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
Carboxypeptidases - metabolism
Cathepsin A
Cell Line
Cysteine Endopeptidases - metabolism
Enzyme Stability
Liver Neoplasms, Experimental - enzymology
Multienzyme Complexes - metabolism
Mutagenesis, Site-Directed
Ornithine Decarboxylase - chemistry
Ornithine Decarboxylase - genetics
Ornithine Decarboxylase - metabolism
Proteasome Endopeptidase Complex
Protein Processing, Post-Translational
Rats
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
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Summary:The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3180879