Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin

Ferreira, Glória C.; Dailey, Harry A.

doi:10.1042/bj2500597

Cited by 46 publications

(20 citation statements)

References 26 publications

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“…Tetrapyrroles have been proposed as signaling molecules that convey information about the status of chloroplasts to the rest of the cell, although the mechanisms involved in tetrapyrrole signaling remain unknown (Strand et al, 2003;Strand, 2004). In this regard, it is noteworthy that the PPO reaction produces 3 mol of hydrogen peroxide (H 2 O 2 ) per mol of protoporphyrinogen IX oxidized (Ferreira and Dailey, 1988). Whereas in the chloroplasts the H 2 O 2 would be quickly dissipated by the abundant stromal AXP, in the mitochondria the H 2 O 2 might be able to participate in peroxide signaling, which has been reported to be involved in regulating a diverse range of responses including development, stress acclimation, and programmed cell death (Gechev and Hille, 2005).…”

Section: Discussionmentioning

confidence: 99%

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

et al. 2005

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Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.

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Section: Discussionmentioning

confidence: 99%

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

et al. 2005

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“…mitochondrial membrane and requires molecular oxygen for catalysis to proceed [3][4][5]. The human, Bacillus subtilis, Myxococcus xanthus, Aquifex aeolicus, and mouse enzymes have previously been cloned, overexpressed in Escherichia coli, and purified to homogeneity [6][7][8][9][10].…”

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confidence: 99%

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Shepherd

Dailey

2005

Analytical Biochemistry

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A continuous spectro fluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless non fluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent K m (K app ) values for protoporphyrinogen IX were measured as 3.8± 0.3, 3.6± 0.5, and 1.0± 0.1 μM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The K i for acifluorfen diphenylether herbicide,was measured as 0.53μM enzyme.Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h -1 /mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min -1 , representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.

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“…It is also hard to assume that relatively mild heat treatment (42°C) directly excites PpIX to generate ROS. One possibility is that H 2 O 2 , generated as a by-product of PpIX biosynthesis by protoporphyrinogen oxidase, 31) is responsible for generating hydroxyl radical via Fenton reaction. However, our observation that PpIX can directly enhance the cell death under thermal stress suggests that PpIX itself, not a byproduct of PpIX biosynthesis, is responsible for the ROS generation and resulting cell death under thermal stress.…”

Section: Discussionmentioning

confidence: 99%

5-aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines

Chibazakura

Toriyabe

Fujii

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation.

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Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin

Cited by 46 publications

References 26 publications

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

5-aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines

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