scholarly journals Distribution Patterns of Ornithine Decarboxylase in Cells and Tissues: Facts, Problems, and Postulates

2002 ◽  
Vol 50 (9) ◽  
pp. 1143-1160 ◽  
Author(s):  
Raymond G. Schipper ◽  
Albert A.J. Verhofstad
Keyword(s):  
Ornithine Decarboxylase ◽  
Cellular Localization ◽  
Distribution Patterns ◽  
Cellular Level ◽  
Physiological Status ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
And Function ◽  
In Cells

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Increased polyamine levels are required for growth, differentiation, and transformation of cells. In situ detection of ODC in cells and tissues has been performed with biochemical, enzyme cytochemical, immunocytochemical, and in situ hybridization techniques. Different localization patterns at the cellular level have been described, depending on the type of cells or tissues studied. These patterns varied from exclusively cytoplasmic to both cytoplasmic and nuclear. These discrepancies can be partially explained by the (lack of) sensitivity and/or specificity of the methods used, but it is more likely that (sub)cellular localization of ODC is cell type-specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Intracellular translocation of ODC may be a prerequisite for its regulation and function.

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.

scholarly journals Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins

1999 ◽  
Vol 10 (12) ◽  
pp. 4299-4310 ◽  
Author(s):  
Mary M. Pomidor ◽  
Rebecca Cimildoro ◽  
Bien Lazatin ◽  
Ping Zheng ◽  
James A. Gurr,· ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Subcellular Fractionation ◽  
Cytochalasin D ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Polyamine Biosynthesis ◽  
Mechanical Disruption ◽  
Confocal Immunofluorescence ◽  
Normal Human

Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [32P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton.

scholarly journals Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA

1996 ◽  
Vol 314 (1) ◽  
pp. 241-248 ◽  
Author(s):  
Anthony J. MICHAEL ◽  
Judith M. FURZE ◽  
Michael J. C. RHODES ◽  
Daniel BURTIN
Keyword(s):  
Ornithine Decarboxylase ◽  
Pseudomonas Syringae ◽  
Datura Stramonium ◽  
Functional Identification ◽  
Polyamine Biosynthesis ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Degenerate Oligonucleotide ◽  
Key Enzyme ◽  
Solanaceous Plant

A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription–PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC route to putrescine is present.

scholarly journals Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth

1994 ◽  
Vol 304 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Y Murakami ◽  
S Matsufuji ◽  
Y Miyazaki ◽  
S Hayashi
Keyword(s):  
Cell Growth ◽  
Ornithine Decarboxylase ◽  
Polyamine Metabolism ◽  
Decarboxylase Activity ◽  
Inhibitory Protein ◽  
Polyamine Biosynthesis ◽  
Rapid Degradation ◽  
Physiological Importance ◽  
Key Enzyme

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. It is a short-lived protein and negatively regulated by its products, polyamines. Its degradation is accelerated by the binding of antizyme, an ODC-inhibitory protein induced by polyamines. To evaluate the physiological importance of antizyme we examined the effect of forced expression of antizyme on cellular ODC and polyamine levels and cell growth. Antizyme almost completely abolished the induction of ODC by growth stimuli. This may have been caused by antizyme-induced rapid degradation of newly synthesized ODC, since the half-life of ODC complexes with antizyme was less than 5 min. Forced expression of antizyme caused reductions of cellular putrescine and spermidine levels, and inhibited cell growth, which was partially restored by the addition of putrescine. These observations suggested a critically important role of antizyme in polyamine metabolism.

scholarly journals A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor

2017 ◽  
Vol 22 (7) ◽  
pp. 906-914 ◽  
Author(s):  
Mohamed Nilam ◽  
Philip Gribbon ◽  
Jeanette Reinshagen ◽  
Kathrin Cordts ◽  
Edzard Schwedhelm ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
High Throughput Screening ◽  
Enzymatic Reaction ◽  
Epigallocatechin Gallate ◽  
Cancer Chemoprevention ◽  
Decarboxylase Activity ◽  
Label Free ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Formed Product

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M−1) than to the substrate L-ornithine (340 M−1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s−1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z′ factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

scholarly journals The ornithine decarboxylase gene of Caenorhabditis elegans: cloning, mapping and mutagenesis.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 517-525 ◽  
Author(s):  
M Macrae ◽  
R H Plasterk ◽  
P Coffino
Keyword(s):  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
In Vitro Translation ◽  
In Vitro Assays ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis ◽  
Eukaryotic Species ◽  
Key Enzyme ◽  
Stage 1

Abstract The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 422 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5' RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved stage 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory.

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.

scholarly journals Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells

1984 ◽  
Vol 221 (2) ◽  
pp. 483-488 ◽  
Author(s):  
P Seppänen ◽  
R Fagerström ◽  
L Alhonen-Hongisto ◽  
H Elo ◽  
P Lumme ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Parent Compound ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
In Cells

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1′-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.

Organization and Function of Stress Fibers in Cells in Vitro and in Situ

1984 ◽  
pp. 83-137 ◽  
Author(s):  
H. Randolph Byers ◽  
Glenn E. White ◽  
Keigi Fujiwara
Keyword(s):  
Stress Fibers ◽  
And Function ◽  
In Cells
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