scholarly journals Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine

2003 ◽  
Vol 375 (2) ◽  
pp. 465-470 ◽  
Author(s):  
Ning QU ◽  
Natalia A. IGNATENKO ◽  
Phillip YAMAUCHI ◽  
David E. STRINGER ◽  
Corey LEVENSON ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Human Colon ◽  
Common Mechanism ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Polyamine Biosynthesis ◽  
Irreversible Reaction ◽  
Kd Values

Racemic difluoromethylornithine (d/l-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. d/l-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either l- or d-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (KD) values for the formation of enzyme–inhibitor complexes were 28.3±3.4, 1.3±0.3 and 2.2±0.4 μM respectively for d-, l- and d/l-DFMO. The differences in these KD values were statistically significant (P<0.05). The inhibitor inactivation constants (Kinact) for the irreversible step were 0.25±0.03, 0.15±0.03 and 0.15±0.03 min−1 respectively for d-, l- and d/l-DFMO. These latter values were not statistically significantly different (P>0.1). d-DFMO was a more potent inhibitor (IC50~7.5 μM) when compared with d-ornithine (IC50~1.5 mM) of ODC-catalysed l-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either l- or d-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme–inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for l-DFMO when compared with d-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the l- and d-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the α-substituent of the inhibitor. The d-enantiomer may have advantages, such as decreased normal tissue toxicity, over l- or d/l-DFMO in some clinical applications.

scholarly journals Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity

1977 ◽  
Vol 166 (1) ◽  
pp. 81-88 ◽  
Author(s):  
A E Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Pyridoxal Phosphate ◽  
Vitamin B ◽  
Decarboxylase Activity ◽  
Deficient Diet ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Ornithine Decarboxylase Activity

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1′-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.

Cu2+-induced modification of the kinetics of Aβ(1-42) channels

2003 ◽  
Vol 285 (4) ◽  
pp. C873-C880 ◽  
Author(s):  
Randa Bahadi ◽  
Peter V. Farrelly ◽  
Bronwyn L. Kenna ◽  
Cyril C. Curtain ◽  
Colin L. Masters ◽  
...  
Keyword(s):  
Amyloid Β ◽  
Chelating Agent ◽  
Common Mechanism ◽  
Amyloid Β Peptide ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Channel Inhibition ◽  
Low Concentrations ◽  
Redox Agents ◽  
In Cis

We found that the amyloid β peptide Aβ(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu2+ or biological redox agents that have been reported to mediate Aβ(1-42) toxicity. The Aβ(1-42)-formed cation channel was inhibited by Cu2+ in cis solution ([Cu2+] cis) in a voltage- and concentration-dependent manner between 0 and 250 μM. The [Cu2+] cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 μM [Cu2+] cis and partially reversible at 250 μM [Cu2+] cis. The inhibitory effects of [Cu2+] cis between 50 and 250 μM on the channel could not be reversed with addition of Cu2+-chelating agent clioquinol (CQ) at concentrations between 64 and 384 μM applied to the cis chamber. The effects of 200-250 μM [Cu2+] cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 μM [CQ] cis. The kinetic analysis of the data indicate that Cu2+-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu2+ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu2+-binding site of the Aβ(1-42)-formed channels is modulated with Cu2+ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu2+ modulation of Aβ and PrP channel proteins linked to neurodegenerative diseases.

scholarly journals The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 740-745 ◽  
Author(s):  
E Niskanen ◽  
A Kallio ◽  
PP McCann ◽  
DG Baker
Keyword(s):  
Ornithine Decarboxylase ◽  
Colony Formation ◽  
Calf Serum ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Host Animal ◽  
Polyamine Biosynthesis ◽  
Diffusion Chambers

Abstract Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2–4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

scholarly journals A study of GDP binding to purified thermogenin protein from brown adipose tissue

1988 ◽  
Vol 251 (2) ◽  
pp. 385-389 ◽  
Author(s):  
R R French ◽  
M G Gore ◽  
D A York
Keyword(s):  
Adipose Tissue ◽  
Fluorescence Quenching ◽  
Equilibrium Dialysis ◽  
Fluorescence Emission ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Brown Adipose ◽  
Kd Values ◽  
Protonated Forms ◽  
Nucleoside Monophosphates

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.

scholarly journals Diamine-induced inhibition of liver ornithine decarboxylase

1979 ◽  
Vol 177 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Arja Kallio ◽  
Monica Löfman ◽  
Hannu Pösö ◽  
Juhani Jänne
Keyword(s):  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Decarboxylase Activity ◽  
Enzyme Protein ◽  
Intracellular Degradation ◽  
Ornithine Decarboxylase Activity ◽  
Normal Rat

Re!peated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ‘antienzymes’ [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858–1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme–inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

Growth hormone inhibition of lipogenesis in sheep adipose tissue: requirement for gene transcription and polyamines

1994 ◽  
Vol 142 (2) ◽  
pp. 235-243 ◽  
Author(s):  
C A Borland ◽  
M C Barber ◽  
M T Travers ◽  
R G Vernon
Keyword(s):  
Growth Hormone ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Ornithine Decarboxylase ◽  
Gene Transcription ◽  
Actinomycin D ◽  
Total Activity ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis

Abstract The chronic inhibitory effect of growth hormone (GH) on lipogenesis in sheep adipose tissue explants was investigated in an in vitro tissue culture system. In the absence of other hormones, GH caused a decrease in the rate of lipogenesis after 6 h of culture. In contrast, when lipogenesis was stimulated by the presence of insulin plus dexamethasone, GH again decreased lipogenesis but after a lag of at least 12 h. Actinomycin D, an inhibitor of gene transcription, prevented the effect of GH on lipogenesis in both the absence and presence of insulin plus dexamethasone. Actinomycin D added to tissue previously incubated for 6 h in the presence of GH alone prevented further decline in lipogenesis over the next 5 h, suggesting that transcription of a short-lived mediator protein is required for the GH effect to occur. An increase in ornithine decarboxylase activity was detected in explants exposed to GH, reaching a peak after 12 h incubation; this was prevented by actinomycin D. Methylglyoxal bis-(guanylhydrazone), an inhibitor of polyamine biosynthesis, partially alleviated the effect of GH on lipogenesis; this was reversed by addition of spermidine. However, spermidine did not reverse the effects of actinomycin D, implicating a short-lived protein in addition to ornithine decarboxylase in the action of GH. In the absence of other hormones GH had no effect on either the expressed (initial) or total activity of acetyl-CoA carboxylase, but GH prevented the increase in both expressed and total activities of the enzyme induced by insulin plus dexamethasone. Varying lipolysis and fatty acid accumulation in adipose tissue by addition of adenosine deaminase plus indomethacin or bovine serum albumin to the culture medium had no effect on lipogenesis and these agents partly alleviated GH inhibition of lipogenesis. No effect of GH was found on the amount of glycerol released by cultured tissue. GH also had no effect on fatty acid esterification. Thus the chronic inhibitory effects of GH on lipogenesis involve a protein with a very short half-life. The effect also requires polyamines but does not appear to involve changes in fatty acid concentrations in the cell. In addition GH appears to inhibit lipogenesis and to antagonise insulin-stimulation of lipogenesis by different mechanisms. Journal of Endocrinology (1994) 142, 235–243

Polyamine-dependent growth and calmodulin-regulated induction of ornithine decarboxylase

1989 ◽  
Vol 256 (2) ◽  
pp. G342-G348
Author(s):  
D. D. Ginty ◽  
E. R. Seidel
Keyword(s):  
Epithelial Cells ◽  
Ornithine Decarboxylase ◽  
Thymidine Incorporation ◽  
Calf Serum ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Polyamine Biosynthesis ◽  
Calmodulin Antagonist ◽  
Serum Stimulation ◽  
Dependent Growth

The proliferation of cultured gastrointestinal crypt epithelial cells (IEC-6) and the role of calcium in polyamine biosynthesis were examined after serum stimulation of quiescent cells. Ornithine decarboxylase (ODC) activity was high when cells grew in 5% fetal calf serum (FCS) and dropped to nearly nondetectable levels when cells reached contact inhibition of growth. Polyamines appeared to be necessary for the proliferation of these cells, as growth was completely inhibited by the addition of 5 mM difluoromethylornithine, a specific inhibitor of ODC, to the media. This effect was reversed by 10 microM putrescine. Serum deprivation of preconfluent cells resulted in a fall in ODC activity. Readdition of serum led to an increase in ODC activity, which peaked at 4 h after addition and preceded both putrescine accumulation and [3H]thymidine incorporation into acid-precipitable material. Furthermore, readdition of serum to serum-deprived cells resulted in an approximately twofold increase in the level of free, ionized, intracellular Ca2+ as measured spectrophotometrically by monitoring fura-2 fluorescence. Inhibition of calmodulin-mediated processes with N-(6-aminohexyl)-5-chloro-1-naphthalelesulfonamide (W-7), a calmodulin antagonist, inhibited the serum-stimulated induction of ODC in a dose-dependent manner, with an IC50 of approximately 10 microM. Similar results were obtained with trifluoperazine. Lastly, 30 microM W-7 completely inhibited serum-stimulated [3H]thymidine incorporation into acid-precipitable material. These data demonstrate that polyamine biosynthesis and subsequent DNA synthesis after serum refeeding of cells is regulated by a Ca2+ activated, calmodulin-dependent process. Furthermore, the production of polyamines is essential for normal proliferation of these epithelial cells in culture.

The Effect of Parathyroid Hormone (1-34) on Cyclic AMP Level, Ornithine Decarboxylase Activity, and Glycosaminoglycan Synthesis of Chondrocytes from Mandibular Condylar Cartilage, Nasal Septal Cartilage, and Spheno-occipital Synchondrosis in Culture

1987 ◽  
Vol 66 (1) ◽  
pp. 84-87 ◽  
Author(s):  
T. Takano ◽  
M. Takigawa ◽  
E. Shirai ◽  
K. Nakagawa ◽  
M. Sakuda ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Ornithine Decarboxylase ◽  
Septal Cartilage ◽  
Decarboxylase Activity ◽  
Condylar Cartilage ◽  
Polyamine Biosynthesis ◽  
Glycosaminoglycan Synthesis ◽  
Mandibular Condylar Cartilage ◽  
Nasal Septal Cartilage

Previously, we reported methods for isolating chondrocytes from the craniofacial complex and their culture in vitro. The response of these chondrocyte cultures to bovine parathyroid hormone (1—34) (PTH) has now been investigated. PTH stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype in cultured chondrocytes isolated from mandibular condylar cartilage (MCC), nasal septal cartilage (NSC), and spheno-occipital synchondrosis (SOS). These stimulations of GAG synthesis by PTH were dose-dependent. PTH also increased accumulation of cyclic AMP (cAMP) and the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis. However, PTH did not stimulate DNA synthesis. The increases in the cAMP level, ODC activity, and GAG synthesis after addition of PTH (10-7 mol/L) were greatest in MCCchondrocytes and least in NSC-chondrocytes. The difference in the responses to PTH of these three types of chondrocytes may reflect differences of the characteristics of these cells in vivo.

Sign in / Sign up

Export Citation Format

Share Document