scholarly journals Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli

1981 ◽  
Vol 200 (1) ◽  
pp. 69-75 ◽  
Author(s):  
A Kallio ◽  
P P McCann
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Arginine Decarboxylase ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Irreversible Inhibitor ◽  
E Coli ◽  
Ornithine Decarboxylase Activity

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.

scholarly journals DL-α-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 771-775 ◽  
Author(s):  
A Kallio ◽  
P P McCann ◽  
P Bey
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Arginine Decarboxylase ◽  
Biosynthetic Enzyme ◽  
Irreversible Inhibitor ◽  
E Coli

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.

Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli

1985 ◽  
Vol 5 (1) ◽  
pp. 161-166
Author(s):  
W A Fonzi ◽  
P S Sypherd
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Ornithine Decarboxylase ◽  
Blot Analysis ◽  
Single Copy ◽  
Decarboxylase Activity ◽  
E Coli ◽  
Ornithine Decarboxylase Activity ◽  
Reduced Frequency ◽  
Diploid Cells

Diploid cells of Saccharomyces cerevisiae homozygous for the spe1A mutation, which eliminates ornithine decarboxylase activity, were found to sporulate at a greatly reduced frequency in the absence of polyamines. Plasmids which complement the spe1A mutation were isolated by their ability to restore sporulation competence to these cells. Three distinct plasmids were isolated. Each plasmid insert overlapped the same 8.0-kilobase region, and each plasmid restored ornithine decarboxylase activity to spe1A mutants. These plasmids also conferred ornithine decarboxylase activity to Escherichia coli EWH319 from which the ornithine decarboxylase gene is deleted. The plasmid-encoded activity expressed in E. coli resembled S. cerevisiae ornithine decarboxylase in its kinetic characteristics, indicating that the yeast ornithine decarboxylase gene was cloned. Southern blot analysis suggested that ornithine decarboxylase is a single-copy gene in S. cerevisiae. A single 2.1-kilobase transcript was demonstrated by Northern blot analysis.

scholarly journals Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli.

1985 ◽  
Vol 5 (1) ◽  
pp. 161-166 ◽  
Author(s):  
W A Fonzi ◽  
P S Sypherd
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Ornithine Decarboxylase ◽  
Blot Analysis ◽  
Single Copy ◽  
Decarboxylase Activity ◽  
E Coli ◽  
Ornithine Decarboxylase Activity ◽  
Reduced Frequency ◽  
Diploid Cells

Diploid cells of Saccharomyces cerevisiae homozygous for the spe1A mutation, which eliminates ornithine decarboxylase activity, were found to sporulate at a greatly reduced frequency in the absence of polyamines. Plasmids which complement the spe1A mutation were isolated by their ability to restore sporulation competence to these cells. Three distinct plasmids were isolated. Each plasmid insert overlapped the same 8.0-kilobase region, and each plasmid restored ornithine decarboxylase activity to spe1A mutants. These plasmids also conferred ornithine decarboxylase activity to Escherichia coli EWH319 from which the ornithine decarboxylase gene is deleted. The plasmid-encoded activity expressed in E. coli resembled S. cerevisiae ornithine decarboxylase in its kinetic characteristics, indicating that the yeast ornithine decarboxylase gene was cloned. Southern blot analysis suggested that ornithine decarboxylase is a single-copy gene in S. cerevisiae. A single 2.1-kilobase transcript was demonstrated by Northern blot analysis.

scholarly journals Involvement of calcium ions in the activation of ornithine decarboxylase by isoprenaline evaluated ‘in situ’ in the perfused rat heart

1983 ◽  
Vol 212 (1) ◽  
pp. 241-243 ◽  
Author(s):  
C Guarnieri ◽  
F Flamigni ◽  
C Muscari ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Calcium Ions ◽  
Decarboxylase Activity ◽  
Ionophore A23187 ◽  
Perfusion Medium ◽  
Perfused Rat Heart ◽  
Ornithine Decarboxylase Activity ◽  
Rat Hearts ◽  
Perfused Hearts

Ornithine decarboxylase activity evaluated during the perfusion of isolated rat hearts by a method ‘in situ’ was rapidly increased when the hearts were infused with isoprenaline (isoproterenol). Omission of Ca2+ from the perfusion medium or the administration of verapamil to the perfused hearts decreased the isoprenaline-stimulated ornithine decarboxylase activity, whereas a marked stimulatory effect was registered when the hearts were perfused with the Ca2+ ionophore A23187.

Biosynthetic ornithine and arginine decarboxylases: correlation of rates of synthesis with activities in Escherichia coli during exponential growth and following nutritional shift-up

1982 ◽  
Vol 28 (8) ◽  
pp. 945-950
Author(s):  
Stephen M. Boyle ◽  
Kazuo Adachi
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ornithine Decarboxylase ◽  
Biological Activities ◽  
Decarboxylase Activity ◽  
Biosynthetic Enzyme ◽  
Guanosine Tetraphosphate ◽  
E Coli ◽  
K 12

Whether guanosine tetraphosphate (ppGpp) has a role in the regulation of the putrescine biosynthetic enzyme, ornithine decarboxylase, in Escherichia coli is controversial. Different laboratories have reported either direct or indirect correlations between ppGpp levels and ornithine decarboxylase activity using different in vivo conditions. In this report, using conditions in vivo to modulate ppGpp levels, experiments are described which bear on the controversy. The rates of synthesis and biological activities of the biosynthetic ornithine and arginine decarboxylases (ODC and ADC) were measured in E. coli K-12 during experimental growth and during nutritional shift-up. There were good correlations between changes in their respective activities and the rates of synthesis of these enzymes during steady state or shift-up. ODC activity or rate of synthesis changed directly in concert with ppGpp levels, while ADC activity or rate of synthesis changed inversely with ppGpp levels. These observations support the contention that ppGpp does not inhibit ODC activity.

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

1982 ◽  
Vol 206 (2) ◽  
pp. 311-318 ◽  
Author(s):  
James E. Seely ◽  
Hannu Pösö ◽  
Anthony E. Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Pyridoxal Phosphate ◽  
Mouse Kidney ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Ornithine Decarboxylase Activity ◽  
Mouse Tissues ◽  
Mammalian Tissues ◽  
Maximal Induction

The binding of α-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 3μm-difluoromethyl[5-14C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having Mr about 100000 and subunit Mr 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[5-14C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-14C]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 α-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 α-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.

scholarly journals dl-α-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of dl-α-difluoromethylarginine to dl-α-difluoromethylornithine

1988 ◽  
Vol 255 (1) ◽  
pp. 197-202 ◽  
Author(s):  
R D Slocum ◽  
A J Bitonti ◽  
P P McCann ◽  
R P Feirer
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Selective Inhibition ◽  
Arginine Decarboxylase ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Hydrolysis Of ◽  
Analogous Mechanism

DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [(3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

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