scholarly journals Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation.

1992 ◽  
Vol 12 (5) ◽  
pp. 2178-2185 ◽  
Author(s):  
L Ghoda ◽  
D Sidney ◽  
M Macrae ◽  
P Coffino
Keyword(s):  
Amino Acids ◽  
Ornithine Decarboxylase ◽  
Distinct Region ◽  
Carboxy Terminus ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Reticulocyte Lysate ◽  
Carboxy Terminal

Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.

Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation

1992 ◽  
Vol 12 (5) ◽  
pp. 2178-2185
Author(s):  
L Ghoda ◽  
D Sidney ◽  
M Macrae ◽  
P Coffino
Keyword(s):  
Amino Acids ◽  
Ornithine Decarboxylase ◽  
Distinct Region ◽  
Carboxy Terminus ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Reticulocyte Lysate ◽  
Carboxy Terminal

Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.

Complementation of a polyamine-deficient Escherichia coli mutant by expression of mouse ornithine decarboxylase

1987 ◽  
Vol 7 (1) ◽  
pp. 564-567
Author(s):  
M Macrae ◽  
P Coffino
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Coli Mutant ◽  
Terminal Amino ◽  
High Level ◽  
Carboxy Terminal ◽  
Exogenous Source

Mouse ornithine decarboxylase (ODCase) cDNA was expressed at a high level in an Escherichia coli mutant deficient in polyamine biosynthesis. The expression of mouse ornithine decarboxylase relieved the dependence of the mutant on an exogenous source of polyamines, presumably by providing putrescine, the product of the enzyme. The effect on the enzymatic activity of deletions that removed carboxy-terminal amino acids of the protein was determined.

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.

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 The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity.

1994 ◽  
Vol 14 (4) ◽  
pp. 2675-2685 ◽  
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
M Hirano ◽  
L T Williams
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Specific Interaction ◽  
Sh2 Domains ◽  
Catalytically Active ◽  
Functional Complex ◽  
Src Homology ◽  
Carboxy Terminal

Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and p110, function as localizing and catalytic subunits, respectively. Using recombinant p85 and p110 molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in p110 which directs the binding to p85. We demonstrate that the interactive domains in p85 and p110 are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and p110 mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length p110 molecule is catalytically active, whereas p110 alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of p110 were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the p110 catalytic subunit, is an important component of the catalytic complex.

Kanamycin Inhibits Cochlear-Renal ODC in Neonatal Rats

1992 ◽  
Vol 107 (4) ◽  
pp. 501-510 ◽  
Author(s):  
Andrew T. Lyos ◽  
William E. Winter ◽  
Charles M. Henley
Keyword(s):  
Organ Of Corti ◽  
Lateral Wall ◽  
Inhibitory Effect ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Old Rats ◽  
In Vivo Effects ◽  
The Brain

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is important in development and regeneration. We hypothesize that aminoglycoside inhibition of ODC mediates developmental hypersensitivity to aminoglycoside ototoxicity. Kanamycin effects on ODC activity (decarboxylation of ornithine) in vitro were determined in the postmitochondriai fraction of cochlear and renal homogenates from 11-day-old rats. Kanamycin inhibited cochlear and renal ODC by an uncompetitive mechanism. For the cochlear enzyme, the inhibitor constant (Ki) for kanamycin was 99 ± 25 (μmol/L; for the renal enzyme, the Ki = 1.5 ± 0.1 mmol/L. In vivo effects of kanamycin on cochlear, renal, brain ODC activity were determined in rats treated with kanamycin (400 mg/kg/day, intramuscularly) or saline during postnatal days 11 through 20, the hypersensitive period for ototoxicity. Rats were killed on postnatal days 12,14,16, and 20 and ODC was assayed. Kanamycin significantly inhibited ODC in the lateral wall-organ of Corti and kidney (ANOVA α = 0.05), but had no effect on cochlear nerve and no consistent inhibitory effect in the brain. These results suggest that ODC is a potential target of kanamycin in susceptible tissues and may be a contributing factor in developmental sensitivity to the drug by inhibiting repair and developmental processes mediated by ODC.

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

2011 ◽  
Vol 301 (1) ◽  
pp. E172-E179 ◽  
Author(s):  
Nicole K. L. Lee ◽  
Jarrod P. J. Skinner ◽  
Jeffrey D. Zajac ◽  
Helen E. MacLean
Keyword(s):  
Skeletal Muscle ◽  
Androgen Receptor ◽  
Ornithine Decarboxylase ◽  
Cell Number ◽  
Male Mice ◽  
Differentiation Markers ◽  
Polyamine Biosynthesis ◽  
Myoblast Proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C2C12 and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C2C12 myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C2C12 myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.

scholarly journals Changes in Liver Gene Expression of Azin1 Knock-out Mice

2010 ◽  
Vol 65 (7-8) ◽  
pp. 519-527 ◽  
Author(s):  
Tao Wan ◽  
Yuan Hu ◽  
Ailong Huang ◽  
Ken-ichi Yamamura ◽  
Hua Tang
Keyword(s):  
Ornithine Decarboxylase ◽  
Regulatory Protein ◽  
Fold Change ◽  
Oligonucleotide Array ◽  
Wild Type ◽  
Polyamine Biosynthesis ◽  
Knock Out ◽  
Knock Out Mice

The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change ≥ 2) and 1466 downregulated genes (fold change ≤ 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

scholarly journals Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase

2007 ◽  
Vol 409 (1) ◽  
pp. 187-192 ◽  
Author(s):  
Kristiina Kanerva ◽  
Laura T. Mäkitie ◽  
Anna Pelander ◽  
Marja Heiskala ◽  
Leif C. Andersson
Keyword(s):  
Ornithine Decarboxylase ◽  
Arginine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Vitro Binding ◽  
In Vitro Binding Assay ◽  
Rate Limiting ◽  
Two Hybrid ◽  
Hybrid Screening

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1–3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.

scholarly journals Amine-specificity of the inactivating ornithine decarboxylase modification in Physarum polycephalum

1982 ◽  
Vol 205 (3) ◽  
pp. 551-557 ◽  
Author(s):  
J L A Mitchell ◽  
G K Mitchell ◽  
D D Carter
Keyword(s):  
Ornithine Decarboxylase ◽  
Translational Control ◽  
Feedback Mechanism ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis ◽  
Polyamine Analogues ◽  
Ornithine Decarboxylase Activity ◽  
Sensitivity Studies

The enzyme catalysing the polyamine-stimulated modification of Physarum ornithine decarboxylase in vivo was partially purified and its activity on purified ornithine decarboxylase was examined with respect to its specificity for various amines. Spermidine, spermine and several polyamine analogues strongly promoted this reaction in vitro (apparent Km in the 0.1-0.5 mM range), whereas putrescine (apparent Km 5.33 mM) and several related diamines were not nearly as effective. In agreement with this, sensitivity studies performed in vivo also suggested that cellular spermidine, and not putrescine, is critical in modulating ornithine decarboxylase activity by this post-translational control. Unlike putrescine, or other diamines, 1,3-diaminopropane demonstrated a functional similarity to the polyamines in stimulating this reaction. This study has demonstrated a method whereby non-physiological amines capable of depressing ornithine decarboxylase activity by this natural feedback mechanism can be readily identified for further evaluation of their potential use in the experimental and medical control of polyamine biosynthesis.

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