New biologically active substances from protozoa and an evaluation of their action]

Novel biologically active substances were obtained from nonpathogenic for humans and free-living protozoa: Crithidia oncopelti, Trypanosoma lewisi, and Astasia longa. There were studied conditions of biosynthesis, composition and biological activity of the total lipid fraction from the protozoa species, sucrose ethers and fatty acids, the latter were isolated from A. longa--(astazilide preparation), reserve beta-1,3 glucan from A. longa (astazian preparation), and fraction of surface glycophospholipids and peptides from Crithidia oncopelti (GLP preparation). Two of three preparations studied (astazilide and GLP) are the complexes of natural substances with certain composition. Conditions of the protozoa cultivation were developed to provide standardization of the complex composition. Division of the complexes into separate components decreased or abolished the biological activity. It was established that the substances studied modify biological reactions, that is exert a systemic effect. They may be used for inhibiting the growth of experimental tumors and preventing the metastatic spreading in tumor-carrier animals. The substances obtained belong to the group of nonspecific stimulators for cellular immunity which can be used in medicine, veterinary medicine, food industry, pharmacologic industry and cosmetology.     

The trypanocidal action of fluorouracil on Crithidia oncopelti in the presence of lipid metabolic modifiers]

Effect of fluorouracil in combination with ascorbate and L-valin, modifiers of lipid metabolism was studied in cultured protozoa Crithidia oncopelti. The inhibitory effect of preparation in a concentration of 100 mcg/ml gradually decreased in the course of cultivation. Its readdition to a 96-hour culture did not increase its effect on protozoa cells. Combined addition of fluorouracil and modifiers (100 mcg/ml each) resulted in insignificant decrease of the cell accumulation in the culture as compared with the effect of fluorouracil alone. When fluorouracil and modifiers were readded to the 96-hour culture, the trypanostatic effect of preparation was 2.5 times enhanced. This enhancement was confirmed by destructive alterations in cell morphology and by the culture lysis by 192 h of protozoa cultivation.     

Ubiquinone system of the form-genusChrysosporium

The ubiquinone (coenzyme Q: Q) system of 17 strains of the form-genusChrysosporium was analyzed by high performance liquid chromatography (HPLC) and found to show a heterogeneous distribution of the major ubiquinone. Q-9, Q-10 or Q-10(H₂) was found to be the major ubiquinone in 3, 9 and 5 strains, respectively. It was further demonstrated that the teleomorphs of the species characterized by Q-9 and Q-10 could be classified into two separate families, Arthrodermataceae (Q-9) and Onygenaceae (Q-10), which were defined within the revised order Onygenales by Currah. Teleomorphs ofChrysosporium species having Q-10(H₂) have not been found. This paper also includes the ubiquinone system of dermatophytes which relate to the form-genusChrysosporium morphologically.     

Nonoxidizable ubiquinol derivatives that are suitable for the study of the ubiquinol oxidation site in the cytochrome bc1 complex

Recent X-ray crystallographic analyses of the mitochondrial cytochrome bc1 complex show ubiquinone binding at the Q(i) site, but attempts to show binding of ubiquinol or ubiquinone at the Q(o) site have been unsuccessful, even though the binding of noncompetitive Q(o) site inhibitors near the putative ubiquinol binding pocket is well established. We speculate that ubiquinol binds transiently to the Q(o) site only when both heme b(L) and the iron sulfur cluster are in the oxidized form, an experimental condition difficult to obtain since ubiquinol will be oxidized once bound to the site. Stable binding at the Q(o) site might be achieved by a nonoxidizable ubiquinol-like compound. For this purpose, the isomers 2,3,4-trimethoxy-5-decyl-6-methyl-phenol (TMDMP) and 2,3,4-trimethoxy-5-methyl-6-decyl-phenol (TMMDP) were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1, 4-benzoquinol (Q0C10) by controlled methylation and separated by TLC and HPLC. The structures of TMDMP and TMMDP were established by 1H-13C-two-dimensional NMR. Both are competitive inhibitors of the cytochrome bc1 complex, with TMDMP being the stronger one. Preliminary results suggest that TMDMP binds tightly enough to make X-ray crystallography of inhibitor-bc1 complex co-crystals feasible. The binding site of TMDMP does not overlap with the binding sites of stigmatellin, MOA-stilbene (MOAS), undecylhydroxydioxobenzothiazole (UHDBT) and myxothaizol.     

Relation of mevalonate synthesis to mitochondrial ubiquinone content and respiratory function in cultured neuroblastoma cells

The consequence of blocking the de novo synthesis of ubiquinone (coenzyme Q) on mitochondrial ubiquinone content and respiratory function was studied in cultured C1300 (Neuro 2A) murine neuroblastoma cells. Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was used to suppress the synthesis of mevalonate, an essential precursor for the isoprenoid side chain of ubiquinone. At a concentration of 25 microM, mevinolin completely inhibited the incorporation of [3H]acetate into ubiquinone, isolated from cell extracts by two-dimensional thin-layer chromatography. Similar results were obtained when [14C]tyrosine was used as a precursor for the quinone ring. Through the use of reverse-phase thin-layer chromatography, it was established that the principal product of the ubiquinone pathway in murine neuroblastoma cells was ubiquinone-9. Inhibition of ubiquinone synthesis for 24h in cells cultured in the presence of 10% fetal calf serum (which contains 0.14 nmol of ubiquinone/ml of serum) resulted in a 40-57% decline in the concentration of ubiquinone in the mitochondria. However, the activities of succinate-cytochrome c reductase and succinate dehydrogenase in whole-cell homogenates or mitochondria were not inhibited. The state 3 and uncoupled rates of respiration, determined by polarographic measurements of oxygen consumption in homogenates and mitochondria, were elevated slightly in the mevinolin-treated cells. The data demonstrate that, although mevalonate synthesis is important for the maintenance of the intramitochondrial ubiquinone pool in cultured cells, major changes in the ubiquinone content of the mitochondria can occur in intact cells without perturbation of respiratory function. However, the coincidence of decreased mitochondrial ubiquinone concentration and the inhibition of cell cycling previously observed in mevinolin-treated cells (Maltese, W.A. (1984) Biochem. Biophys. Res. Commun. 120, 454-460) suggests that the availability of ubiquinone may play a role in the regulation of mitochondrial and cellular proliferation.     

Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis

Abstract The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus.     

Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.     

Characterization of solanesyl and decaprenyl diphosphate synthases in mice and humans

The isoprenoid chain of ubiquinone (Q) is determined by trans-polyprenyl diphosphate synthase in micro-organisms and presumably in mammals. Because mice and humans produce Q9 and Q10, they are expected to possess solanesyl and decaprenyl diphosphate synthases as the determining enzyme for a type of ubiquinone. Here we show that murine and human solanesyl and decaprenyl diphosphate synthases are heterotetramers composed of newly characterized hDPS1 (mSPS1) and hDLP1 (mDLP1), which have been identified as orthologs of Schizosaccharomyces pombe Dps1 and Dlp1, respectively. Whereas hDPS1 or mSPS1 can complement the S. pombe dps1 disruptant, neither hDLP1 nor mDLP1 could complement the S. pombe dLp1 disruptant. Thus, only hDPS1 and mSPS1 are functional orthologs of SpDps1. Escherichia coli was engineered to express murine and human SpDps1 and/or SpDlp1 homologs and their ubiquinone types were determined. Whereas transformants expressing a single component produced only Q8 of E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q9 or Q10, respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long-chain trans-prenyl diphosphate synthases, as estimated by gel-filtration chromatography, indicates that they consist of heterotetramers. Expression in E. coli of heterologous combinations, namely, mSPS1 and hDLP1 or hDPS1 and mDLP1, generated both Q9 and Q10, indicating both components are involved in determining the ubiquinone side chain. Thus, we identified the components of the enzymes that determine the side chain of ubiquinone in mammals and they resembles the S. pombe, but not plant or Saccharomyces cerevisiae, type of enzyme.     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.     

Oxygen toxicity in Astasia

1. Exposure of Astasia longa to oxygen+carbon dioxide (95:5) at atmospheric pressure leads to an inhibition of growth rate and of respiration. Growth resumes at the normal rate as soon as the oxygenation is discontinued, but respiration recovers more slowly. 2. Mitochondria prepared from cells exposed to oxygen+carbon dioxide (95:5) during growth have considerably decreased activities of succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, succinate dehydrogenase and succinate oxidase activities as compared with mitochondria obtained from cells exposed to air+carbon dioxide (95:5). Cytochrome oxidase activity is not appreciably inhibited by exposure of the cells to 95% oxygen. 3. The mitochondrial fraction of Astasia contains rhodoquinone. The rhodoquinone concentration increases in cells exposed to 95% oxygen. The content of ergosterol-containing compounds also increases in the mitochondria of cells exposed to 95% oxygen. There is little change in the ubiquinone content of the mitochondrial fraction. The ubiquinone of Astasia appears to be ubiquinone-45.     

Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b.     

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

Antioxidant action of ubiquinol homologues with different isoprenoid chain length in biomembranes

Ubiquinones (CoQn) are intrinsic lipid components of many membranes. Besides their role in electron-transfer reactions they may act as free radical scavengers, yet their antioxidant function has received relatively little study. The efficiency of ubiquinols of varying isoprenoid chain length (from Q0 to Q10) in preventing (Fe2+ + ascorbate)-dependent or (Fe2+ + NADPH)-dependent lipid peroxidation was investigated in rat liver microsomes and brain synaptosomes and mitochondria. Ubiquinols, the reduced forms of CoQn, possess much greater antioxidant activity than the oxidized ubiquinone forms. In homogenous solution the radical scavenging activity of ubiquinol homologues does not depend on the length of their isoprenoid chain. However in membranes ubiquinols with short isoprenoid chains (Q1-Q4) are much more potent inhibitors of lipid peroxidation than the longer chain homologues (Q5-Q10). It is found that: i) the inhibitory action, that is, antioxidant efficiency of short-chain ubiquinols decreases in order Q1 greater than Q2 greater than Q3 greater than Q4; ii) the antioxidant efficiency of long-chain ubiquinols is only slightly dependent on their concentrations in the order Q5 greater than Q6 greater than Q7 greater than Q8 greater than Q9 greater than Q10 and iii) the antioxidant efficiency of Q0 is markedly less than that of other homologues. Interaction of ubiquinols with oxygen radicals was followed by their effects on luminol-activated chemiluminescence. Ubiquinols Q1-Q4 at 0.1 mM completely inhibit the luminol-activated NADPH-dependent chemiluminescent response of microsomes, while homologues Q6-Q10 exert no effect. In contrast to ubiquinol Q10 (ubiquinone Q10) ubiquinone Q1 synergistically enhances NADPH-dependent regeneration of endogenous vitamin E in microsomes thus providing for higher antioxidant protection against lipid peroxidation. The differences in the antioxidant potency of ubiquinols in membranes are suggested to result from differences in partitioning into membranes, intramembrane mobility and non-uniform distribution of ubiquinols resulting in differing efficiency of interaction with oxygen and lipid radicals as well as different efficiency of ubiquinols in regeneration of endogenous vitamin E.     

Ubiquinone binding, ubiquinone exclusion, and detailed cofactor conformation in a mutant bacterial reaction center

The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.     

Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site

Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).     

Coenzyme Q homologs in parasitic protozoa as targets for chemotherapeutic attack

The central role of coenzyme Q (ubiquinone) in cellular energy metabolism is well established. Recent work has implicated this molecule in a wide range of other cellular functions, including roles in growth control, plasma membrane oxidase and as a cellular antioxidant. In this review, Jayne Ellis presents an overview of the current knowledge of this important cellular component in species of parasitic protozoa, discusses current therapies using its analogs and proposes its potential roles in these organisms.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).