Evidence for the amino-acid sequence of Crithidia fasciculata Cytochrome c555.

Evidence is presented which indicates that the amino acid sequence of cytochrome c555 from Crithidia fasciculata differs at sixteen positions from that of cytochrome c557 from Crithidia oncopelti. 101 residues were identified by dansyl-Edman degradation, carboxypeptidase digestion or considerations of the specificity of trypsin and of these, thirteen were found to be different from C. oncopelti cytochrome c557. The remaining 11 residues found in the amino acid composition of the trypic peptides were aligned on the basis of homology with cytochrome c557 and three further differences are proposed. The total of sixteen amino acid differences is surprising in view of the morphological and biochemical similarities of these organisms, and illustrates the problem of taxonomy of morphologically simple organisms. In both cytochromes only one cysteine residue is involved in the attachment of the protein to the prosthetic group.     

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

A cytochrome c methyltransferase from Crithidia oncopelti.

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

Spliced leader-associated RNA from Crithidia fasciculata contains a structure resembling stem/loop II of U1 snRNA.

In contrast to earlier proposals, recent evidence suggests that trans-spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23,691-23,694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader-associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculata and propose that it may contribute a U1 snRNA-like stem/loop II to the trans-spliceosome.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

Physicochemical properties of two atypical cytochromes c, Crithidia cytochrome c-557 and Euglena cytochrome c-558.

Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic group. Similarly the alkaline isomers of cytochromes c-557 and c-558 give rise to high-spin proteins above pH 11. The alkaline isomerization of cytochrome c-558, involves a pKobs. of 10 and kinetics which do not obey the model of Davis et al. [(1974) J. Biol. Chem. 249, 2624-2632] for horse cytochrome c. It is proposed that a model involving two ionizations, followed by a conformation change, may fit the data. Both cytochromes c-557 and c-558 combine slowly with CO at neutral pH values.     

Cytochrome c-mediated electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase. Kinetic evidence for a mobile cytochrome c pool.

Ubiquinol oxidase has been reconstituted from ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase (Complex IV). The steady-state level of reduction of cytochrome c by ubiquinol-2 varies with the molar ratios of the complexes and with the presence of antimycin in a way that can be quantitatively accounted for by a model in which cytochrome c acts as a freely diffusible pool on the membrane. This model was based on that of Kröger & Klingenberg [(1973) Eur. J. Biochem. 34, 358-368] for ubiquinone-pool behaviour. Further confirmation of the pool model was provided by analysis of ubiquinol oxidase activity as a function of the molar ratio of the complexes and prediction of the degree of inhibition by antimycin.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN.     

Characterization of a novel endonuclease from Crithidia fasciculata.

A new endonuclease activity has been identified in whole cell lysates of the trypanosomatid Crithidia fasciculata. This activity, termed endonuclease A (Endo A), introduces single-strand breaks at highly preferred sites in double stranded DNA substrates Physical analysis of this enzyme indicates that it has a sedimentation coefficient S20,W of 4.9 and a Stokes radius of 59A and thus, a native molecular weight of 125,000 and a frictional coefficient of 1.8. A monomeric structure is suggested for the enzyme based on the recovery of Endo A activity associated with a polypeptide with a molecular weight of 116,000-120,000, following electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Endo A shows an absolute requirement for Mg2+ or Mn2+ and exhibits activity over a broad pH and temperature range, with optimal conditions for activity at pH 8.0 and 30 degrees C.     

Cytochrome c interaction with membranes. Interaction of cytochrome c with isolated membrane fragments and purified enzymes

The midpoint redox potential of cytochrome c and the electron paramagnetic resonance spectra of nitroxide labeled cytochromes c were measured as a function of binding to purified cytochrome c oxidase, cytochrome c peroxidase, cytochrome b₅ and succinate—cytochrome c reductase. The midpoint redox potential of horse heart cytochrome c is lowered in the presence of cytochrome c oxidase and succinate-cytochrome c reductase, but is unchanged in the presence of cytochrome c peroxidase or cytochrome b₅. Further evidence of binding is afforded by an increase in correlation time, T_c, of the spin-labeled cytochrome c at methionine 65 upon binding to cytochrome c peroxidase, cytochrome c oxidase and succinate—cytochrome c reductase. The changes in midpoint redox potential and electron paramagnetic resonance spectrum of the spin-labeled derivative upon binding can either be the consequence of specific interaction leading to formation of ES complexes, or it can be due to nonspecific electrostatic interaction between positively charged groups on cytochrome c and negatively charged groups on the isolated cytochrome preparations.     

Toxicity of 4-nitroquinoline 1-oxide for Crithidia fasciculata.

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cystein, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.