Abstract
The mechanisms of the response of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with isoosmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effecters and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA, respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [S-35]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells, However, it also appears to occur in concert with decreased enzyme inactivation and/or degradation.
Original language | English |
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Pages (from-to) | 401-408 |
Number of pages | 8 |
Journal | Biochemical Journal |
Volume | 348 |
Publication status | Published - 2000 |
Keywords
- glycine
- polyamine biosynthesis
- nutrient
- ODC
- post-translational regulation
- RAT-LIVER
- INDUCTION
- PROTEIN
- EXPRESSION
- TRANSPORT
- ASPARAGINE
- MECHANISMS
- RNA
- DIFFERENTIATION
- STIMULATION