A comparison of ornithine decarboxylases from normal and SV40-transformed 3T3 mouse fibroblasts

Weiss, J.; Lembach, Kenneth J.; Boucek, Robert J.

doi:10.1042/bj1940229

Cited by 13 publications

(8 citation statements)

References 47 publications

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“…On the other hand, the titration with a-(difiuoromethyl) [5-3H] ornithine gives additional information in that the electrophoretic and chromatographic properties of the labeled protein can be determined. As shown in Figure 3, the subunit molecular weight of the ornithine decarboxylase in SV-3T3 cells at all times after stimulation was about 53000, in good agreement with previous estimates of the molecular weight of the rat and mouse enzyme (Pritchard et al, 1981;Persson, 1981;Seely et al, 1982b;Kameji & Hayashi, 1982) and of the purified enzyme from SV-3T3 cells (Weiss et al, 1981). The enzyme was purified by Boucek and colleagues, but even after a 7000-fold purification, the specific activity of their purified enzyme was only 120000 units/mg (Boucek & Lembach, 1977;Weiss et al, 1981) whereas our results suggest that the homogeneous enzyme would have a specific activity of about 1 300000 units/mg.…”

Section: Discussionsupporting

confidence: 91%

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts

Erwin¹,

Seely²,

Pegg³

1983

Biochemistry

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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.

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Section: Discussionsupporting

confidence: 91%

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts

Erwin¹,

Seely²,

Pegg³

1983

Biochemistry

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“…More recently, Kameji et al (1981) have purified rat liver ornithine decarboxylase to approximately the predicted specific activity. The predicted specific activity of the mouse enzyme is 50,umol of C02/min per mg, which is also more than that so far obtained for the enzyme from mouse tissues and cells (Weiss et al, 1981;Persson, 1981), but we have subsequently purified the enzyme from mouse kidney to this value (Seely et al, 1982).…”

Section: Discussionmentioning

confidence: 72%

“…Most of the studies of changes in ornithine decarboxylase levels have been limited to measurements of enzyme activity, although some attemnpts have been made to quantify the amount of enzyme actually present by the use of immunological techniques (H6ltta, 1975;Oben-0306-3283/82/080311-08$01.50/1 © 1982 The Biochemical Society rader & Prouty, 1977;Kallio et al, 1977Kallio et al, , 1979Weiss et al, 1981). Theoretically, such work should provide an unequivocal answer to the question of whether changes in ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein, but, for a variety of reasons, none of these studies is entirely conclusive.…”

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confidence: 99%

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

Seely

Pösö

Pegg

1982

Biochemical Journal

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The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.

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“…ODC activity was assayed by measuring the release of 14 CO 2 from L-[1-14 C]ornithine (70). The cells were washed twice in PBS and lysed in ice-cold buffer containing 0.5% (vol/vol) Triton X-100, 25 mM Tris-HCl (pH 7.5 at 22°C), 0.1 mM EDTA, and 1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

A Role for c-myc in Chemically Induced Renal-Cell Death

Zhan

Cleveland

Stevens

1997

Molecular and Cellular Biology

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A comparison of ornithine decarboxylases from normal and SV40-transformed 3T3 mouse fibroblasts

Cited by 13 publications

References 47 publications

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

A Role for c-myc in Chemically Induced Renal-Cell Death

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