Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells.
The half-life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC-degrading activity. SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse-transcription/polymerase-chain-reaction (RT-PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441-->Trp replacement near the C-terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC-deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild-type ODC activity decreased with a half-life of 1 h. In-vitro-synthesized mutant ODC with the Cys441-->Trp (or Ala) replacement was also stable in a reticulocyte-lysate ODC-degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441-->Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.
Miyazaki
Y
Y
Department of Nutrition, Jikei University School of Medicine, Tokyo, Japan.
Matsufuji
S
S
Murakami
Y
Y
Hayashi
S
S
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line / Q000201:enzymology; D002474:Cell-Free System; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics; D004795:Enzyme Stability; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection
D016372:RNA, Antisense; D012333:RNA, Messenger; D009955:Ornithine Decarboxylase; D003545:Cysteine