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 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.
Mitchell
J L
JL
Department of Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Choe
C Y
CY
Judd
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase