Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme. In the present study. PfPPO has been cloned, expressed and shown to be localized to the mitochondrion by immunofluorescence microscopy. Interestingly, the enzyme has been found to be active only under anaerobic conditions and is dependent on electron transport chain (ETC) acceptors for its activity. The native enzyme present in the parasite is inhibited by the ETC inhibitors, atovaquone and antimycin. Atovaquone, a well known inhibitor of parasite dihydroorotate dehydrogenase, dependent on the ETC, inhibits synthesis of heme as well in P. falciparum culture. A model is proposed to explain the ETC dependence of both the pyrimidine and heme-biosynthetic pathways in P. falciparum. (C) 2010 Elsevier B.V. All rights reserved.

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Brown adipose tissue mitochondria are cytochrome c — subsaturated

The oxidative activity of mitochondria freshly isolated from brown adipose tissue of rats was stimulated two-fold on the addition of small concentrations of exogenous cytochrome c to the reaction medium. Loss of membrane-bound cytochrome c did not occur during isolation of mitochondria. Estimation of the high-affinity binding sites on the organelle membrane indicated that less than a third of these sites remained saturated with cytochrome c. The pigment is thus shown to be a functionally limiting electron transport component in brown adipose tissue.

Destruction Of Rat-Liver Microsomal Cytochrome-P-450 Invitro By A Monoterpene Ketone, Pulegone A Hepatotoxin

Significant destruction (68%) of liver microsomal cytochrome P-450 and homogeneous cytochrome P-450 purified from PB-treated rats is noticed upon incubation with 10 mM pulegone at 37-degrees-C for 30 min. There is also a concomitant loss of heme. The destructive phenomenon does not require metabolic activation of pulegone. The destruction of purified cytochrome P-450 is time-dependent and saturable. Structure-activity studies suggest that an alpha-isopropylidine ketone unit with a methyl positioned para to the isopropylidine group as in pulegone is necessary for the in vitro destruction of cytochrome P-450. SKF-525A at a concentration of 4-mM obliterates the destruction of cytochrome P-450 by pulegone. Experiments with C-14-pulegone suggest that pulegone or its rearranged product binds covalently to the prosthetic heme of cytochrome P-450.

Estradiol - 17β induces polyaromatic hydrocarbon-inducible cytochrome P-450 in chicken liver

A cDNA clone has been isolated from a chicken liver library prepared against messenger RNA isolated after chronic estradiol-17β treatment. The clone, pP-450 IA - 61, has an insert of 900nt and the sequence shows high homology to CYPIA2 subfamily from four other species. A single injection of estradiol-17β to immature chicken results in a striking induction of mRNA hybridizing to labeled pP-450IA - 61. The probe also hybridizes to mRNA induced by 3 — methylcholanthrene in chicken. These results offer direct proof for the similarity in the mode of action at the transcriptional level of polyaromatic hydrocarbons and estrogenic compounds.

Studies on the biosynthesis of cytochrome P-450 in rat liver—A probe with phenobarbital

Cytochrome P-450 has been purified from phenobarbital-treated rat livers to a specific content of 17 nmol/mg protein. The major species purified has a molecular weight of 48,000. Using the purified antibody for the cytochrome P-450 preparation it has been shown that the major product synthesized in vivo and in the homologous cell-free system in vitro is the 48,000 molecular weight species. Poly(A)-containing RNA isolated from phenobarbital-treated animals codes for the synthesis of the 48,000 molecular weight species in the wheat germ and reticulocyte lysate cell-free systems. It is concluded that cytochrome P-450 synthesis does not involve processing of a polyprotein precursor, although certain minor modifications including glycosylation of the primary translation product are not ruled out. Phenobarbital treatment of the animal results in a significant increase in the cytochrome P-450 messenger activity as measured in the wheat germ cell-free system.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Dexamethasone negatively regulates phenobarbitone-activated transcription but synergistically enhances cytoplasmic levels of cytochrome P-450b/e messenger RNA

Dexamethasone has a potentiating effect on phenobarbitone mediated induction of cytochrome P-450b + e mRNAs in adult rat liver. However, the glucocorticoid inhibits phenobarbitone-activated transcription of cytochrome P-450b + e mRNAs by 60-70%. This inhibitory effect is evident in run-off transcription of the endogenous genes as well as in the transcription of an added cloned gene fragment. Dexamethasone inhibits the phenobarbitone-mediated increase in the binding of a transcription factor(s) to the upstream region of the gene as evidenced by gel retardation and Southwestern blot analysis. The glucocorticoid does not stabilize the phenobarbitone-induced polyribosomal cytochrome P-450b + e mRNAs but appears to stabilize the nuclear transcripts. It is proposed that a negative element may mediate the action of dexamethasone at the level of nuclear transcription and stabilization of the nuclear transcript may account for the potentiating effect of the glucocorticoid on phenobarbitone-mediated increase in cytochrome P-450b + e mRNAs in the cytoplasm of the adult rat liver. However, the cytochrome P-450b protein levels are slightly lower in phenobarbitone + dexamethasone treatment than in phenobarbitone-treated liver microsomes.

Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.

Reinstate hydrogen peroxide as the product of alternative oxidase of plant mitochondria

Chill treatment of potato tubers for 8 days induced mitochondrial O-2 consumption by cyanide-insensitive alternative oxidase (AOX). About half of the total O-2 consumption in such mitochondria was found to be sensitive to salicylhydroxamate (SHAM), a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive O-2 consumption by nearly half, and addition at the end of the reaction released half of the O-2 consumed by AOX, both typical of catalase action on H2O2. This reaffirmed that the product of reduction of O-2 by plant AOX was H2O2 as found earlier and not H2O as reported in some recent reviews.

Involvement of Cytochrome P450 in Conferring Chloroquine Resistance to the Malarial Parasite, Plasmodium falciparum

The higher levels of cytochrone P-450 dependent enzyme activities reported earlier are traced to higher levels of cytochrome P-450 (CYPIIB1/B2 like) messenger RNA in the chloroquine resistant than the sensitive strains. The messenger RNA is also induced by phenobarbitone in the sensitive strain. Pretreatment with phenobarbitone affords partial protection to chloroquine toxicity in the sensitive strain and this is not due to a differential accumulation of the drug.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome Image via cytochrome b5 but not to menadione.

Functions Of Cytochrome-C In Regulation Of Electron-Transfer And Protein Folding

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.