Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts

Abstract

Stimulation of confluent serum-starved SV-3T3 cells by serum produces a transient enhancement of ornithine decarboxylase activity which fluctuates at least 20-fold over a 12-h period. The mechanism by which this fluctuation is produced was investigated. Two techniques for assaying the amount of enzyme protein in these cells were utilized: radioimmunoassay and titration with alpha-(difluoromethyl) [5-3H]ornithine. The radioimmunoassay was carried out by using a specific antiserum prepared in rabbits against homogeneous mouse kidney ornithine decarboxylase and by using alpha-(difluoromethyl) [5-3H]ornithine-labeled kidney ornithine decarboxylase as the tracer ligand. An exact correlation between the amount of enzyme protein and the amount of enzyme activity was seen during the rise and fall of ornithine decarboxylase activity after serum stimulation. Similarly, the amount of protein which was labeled covalently by reaction with alpha-(difluoromethyl) [5-3H]ornithine (a specific enzyme-activated irreversible inhibitor) correlated with the enzyme activity. Investigation of the protein labeled in this way by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that no change in the size of the protein which had a molecular weight of 53 000 occurred during this time period. These results indicate the alteration in ornithine decarboxylase activity can be accounted for entirely by changes in the amount of enzyme protein rather than by posttranslational modifications, activators, or inhibitors.

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen‐activated lymphocytes

“…After a 30-min incubation at 37 "C the cells were collected on ice and washed twice with ice-cold phosphate-buffered saline. The procedure for preparing cell extracts has been previously described [16]. Total [35S]methionine incorporation was determined by trichloroacetic acid precipitation of an aliquot of each sample.…”

Gene expression of ornithine decarboxylase in L1210 leukaemia cells exposed to dl-2-difluoromethylornithine in the presence of cadaverine

“…ODC activity was assayed by the method of Janne & Williams-Ashman (1971). Immunoreactive ODC was determined by the method of Erwin et al (1983), by using monospecific antibody raised in rabbits (Isomaa et al, 1983). Genomic DNA was isolated by the method of Blin & Stafford (1976).…”

Role of G-proteins in T cell activation: non-hydrolysable GTP analogues induce early ornithine decarboxylase activity in human T lymphocytes.

“…Omithine decarboxylase (ODC) is the rate-limiting enzyme in the synthesis of polyamines (Janne et al, 1978). Cell growth is invariably associated with high ODC activities and stimulated polyamine metabolism (Kay and Lindsay, 1973;Korpela et al, 1981;Erwin et al, 1983;Persson et al, 1984;Kallio et al, 1977, Haddox et al, 1980. Mitogenic ligands (but not nonmitogenic) induce a 4to 5-fold activation of ODC in T cells within 5-10 mmin (Scott et al, 1984.…”