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

ABSTRACT: 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.

SUBMITTER: Slocum RD

PROVIDER: S-EPMC1135209 | biostudies-other | 1988 Oct

REPOSITORIES: biostudies-other

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Project description:Background:Polyamines stimulate DNA transcription and mRNA translation for protein synthesis in trophectoderm cells, as well as proliferation and migration of cells; therefore, they are essential for development and survival of conceptuses (embryo/fetus and placenta). The ovine conceptus produces polyamines via classical and non-classical pathways. In the classical pathway, arginine (Arg) is transformed into ornithine, which is then decarboxylated by ornithine decarboxylase (ODC1) to produce putrescine which is the substrate for the production of spermidine and spermine. In the non-classical pathway, Arg is converted to agmatine (Agm) by arginine decarboxylase (ADC), and Agm is converted to putrescine by agmatinase (AGMAT). Methods:Morpholino antisense oligonucleotides (MAOs) were designed and synthesized to inhibit translational initiation of the mRNAs for ODC1 and ADC, in ovine conceptuses. Results:The morphologies of MAO control, MAO-ODC1, and MAO-ADC conceptuses were normal. Double knockdown of ODC1 and ADC (MAO-ODC1:ADC) resulted in two phenotypes of conceptuses; 33% of conceptuses appeared to be morphologically and functionally normal (phenotype a) and 67% of the conceptuses presented an abnormal morphology and functionality (phenotype b). Furthermore, MAO-ODC1:ADC (a) conceptuses had greater tissue concentrations of Agm, putrescine, and spermidine than MAO control conceptuses, while MAO-ODC1:ADC (b) conceptuses only had greater tissue concentrations of Agm . Uterine flushes from ewes with MAO-ODC1:ADC (a) had greater amounts of arginine, aspartate, tyrosine, citrulline, lysine, phenylalanine, isoleucine, leucine, and glutamine, while uterine flushes of ewes with MAO-ODC1:ADC (b) conceptuses had lower amount of putrescine, spermidine, spermine, alanine, aspartate, glutamine, tyrosine, phenylalanine, isoleucine, leucine, and lysine. Conclusions:The double-knockdown of translation of ODC1 and ADC mRNAs was most detrimental to conceptus development and their production of interferon tau (IFNT). Agm, polyamines, amino acids, and adequate secretion of IFNT are critical for establishment and maintenance of pregnancy during the peri-implantation period of gestation in sheep.

Project description: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 (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) 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 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 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 alpha-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.

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

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