Characterization of highly purified ornithine decarboxylase from rat heart

Flamigni, Flavio; Guarnieri, Carlo; Caldarera, C. M.

doi:10.1016/0304-4165(84)90168-5

Cited by 27 publications

(11 citation statements)

References 25 publications

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“…Gel filtration analyses revealed that the active protein is about 90 kDa and is thus a homodimer. This is consistent with the quaternary structure of other ODCs [44,45], with the exception of the T. aginalis ODC, which is active as a tetramer [46].…”

Section: Discussionsupporting

confidence: 89%

“…The K m determined for P. redi i us recombinant ODC is similar to those reported for mammalian ODCs [45,47]. α-DFMO was found to have an apparent K i of 34 µM and a τ &!…”

Section: Discussionsupporting

confidence: 76%

“…This corresponds to the results obtained by the sequence analyses of the cDNA and shows that no posttranslational processing of the protein is necessary to obtain catalytically active ODC. The molecular mass of the P. redi i us ODC monomer is very similar to those of other eukaryotes [39,[44][45][46]. Gel filtration analyses revealed that the active protein is about 90 kDa and is thus a homodimer.…”

Section: Discussionmentioning

confidence: 87%

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Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Niemann

Besser

Walter

1996

Biochemical Journal

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A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.

show abstract

Section: Discussionsupporting

confidence: 89%

“…The K m determined for P. redi i us recombinant ODC is similar to those reported for mammalian ODCs [45,47]. α-DFMO was found to have an apparent K i of 34 µM and a τ &!…”

Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 87%

See 1 more Smart Citation

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Niemann

Besser

Walter

1996

Biochemical Journal

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“…hODC might serve as a numerical example: K m values for ornithine of 70 to 90 μM (17, 25) and K d values for PLP of 0. 1 to 0.3 μM have been reported (28–30). The K d value of the noncovalently binding coenzyme moiety of the conjugate inhibitor might be assumed to be almost 100‐fold higher than that for PLP due to the loss of the contribution of the internal aldimine linkage (31, 32).…”

Section: Novel Strategy For Designing Inhibitors Of B6 Enzymesmentioning

confidence: 99%

A novel approach to inhibit intracellular vitamin B₆‐dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase

Christen

Gehring

2011

FASEB j.

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Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 μM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest.

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“…In all PLP-dependent enzymes, the external aldimine, a Schiff base formed between the cofactor PLP and the amino group of the amino acid substrate (ES-PLP), is a common intermediate (19,20). PLP binds with high affinity to PLPdependent enzymes (21). Reduction of the Schiff base produces phosphopyridoxyl-amino acid conjugates, analogs of the covalent intermediates.…”

mentioning

confidence: 99%

Structural requirements for novel coenzyme‐substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

Gehring²

2008

FASEB j.

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Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.

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Characterization of highly purified ornithine decarboxylase from rat heart

Cited by 27 publications

References 25 publications

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

A novel approach to inhibit intracellular vitamin B₆‐dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase

Structural requirements for novel coenzyme‐substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

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Characterization of highly purified ornithine decarboxylase from rat heart

Cited by 27 publications

References 25 publications

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

A novel approach to inhibit intracellular vitamin B6‐dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase

Structural requirements for novel coenzyme‐substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

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A novel approach to inhibit intracellular vitamin B₆‐dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase