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Use of 4-fluoro-L-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway.
- Source :
-
Biochemical pharmacology [Biochem Pharmacol] 1995 Oct 26; Vol. 50 (9), pp. 1433-43. - Publication Year :
- 1995
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Abstract
- The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.
- Subjects :
- Adenosylmethionine Decarboxylase biosynthesis
Animals
Biogenic Polyamines metabolism
CHO Cells
Cell Division drug effects
Cell Membrane Permeability
Chromatography, High Pressure Liquid
Cricetinae
Humans
Kinetics
Leukemia L1210
Melanoma metabolism
Mice
Ornithine analysis
Ornithine pharmacokinetics
Ornithine pharmacology
Ornithine Decarboxylase biosynthesis
Spermidine metabolism
Spermine metabolism
Tumor Cells, Cultured
Biogenic Polyamines biosynthesis
Ornithine analogs & derivatives
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2952
- Volume :
- 50
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Biochemical pharmacology
- Publication Type :
- Academic Journal
- Accession number :
- 7503794
- Full Text :
- https://doi.org/10.1016/0006-2952(95)02037-3