cytochrome-c-t and 4-6-dinitro-o-cresol

iNOS lacks any phosphorylatable residue at its C-terminus despite displaying a 25-residue extension known to block electron transfer and activity. We report that C-terminal deletions of iNOS increased the cytochrome c reduction rate. Moreover, the interaction of the iNOS C-terminus with the PDZ domains of EBP50 or CAP70 resulted not only in augmented reductase activity and greater NO synthesis but also anticipated the formation of the air-stable semiquinone generated after NADPH addition. Hence, the C-terminus of iNOS regulates the activity of the enzyme, albeit, unlike nNOS and eNOS, displacement of the autoinhibitory element occurs upon binding to proteins with PDZ domains.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; Chlorocebus aethiops; COS Cells; Cytochromes c; Dinitrocresols; Electron Transport; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Molecular Sequence Data; NADP; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidation-Reduction; Phosphoproteins; Protein Binding; Sequence Homology, Amino Acid; Sodium-Hydrogen Exchangers; Spectrophotometry

In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.

Topics: Apoptosis; Caspase 3; Cells, Cultured; Cytochromes c; Dinitrocresols; DNA Fragmentation; Glycine max; Herbicides; Mitochondria; Mitochondrial Proteins; Oxidoreductases; Plant Proteins

Topics: Biochemical Phenomena; Biochemistry; Coenzymes; Cytochromes; Cytochromes c; Dinitrocresols; Flavin Mononucleotide; Flavins; NADP; NADPH-Ferrihemoprotein Reductase; Oxidoreductases; Phosphates; Research; Riboflavin

Topics: Coenzymes; Cytochromes c; Dinitrocresols; Flavins; NADH Dehydrogenase; Neurospora; Nitrate Reductase; Nitrate Reductases; Oxidoreductases; Phosphates

Topics: Cytochromes; Cytochromes c; Dinitrocresols; Enzymes; Flavins; Iron