Leishmania tarentolae: A Parallel Isolation of Cytochrome bc1 and Cytochrome c Oxidase

doi:10.1006/expr.2000.4564

Experimental Parasitology

Volume 96, Issue 3, November 2000, Pages 160-167

https://doi.org/10.1006/expr.2000.4564 Get rights and content

Abstract

Horváth, A., Berry, E. A., Huang L., and Maslov, D. A. 2000. Leishmania tarentolae: A parallel isolation of cytochrome bc₁ and cytochrome c oxidase. Experimental Parasitology96, 160–167. A rapid and simple method which allowed for a parallel isolation of cytochrome c reductase (cytochrome bc₁ ) and cytochrome c oxidase from kinetoplast-mitochondria of Leishmania tarentolae was developed. The method involved the lysis of kinetoplasts with dodecyl maltoside in the presence of 260 mM NaCl, followed by purification of bc₁ complexes on DEAE–sepharose CL-6B. The oxidase which was found in the flow-through fractions of the first chromatographic step was diluted and then repurified on a similar DEAE–sepharose column. The investigated properties of the isolated cytochrome c oxidase and reductase, such as their absolute and difference spectrum absorption maxima, heme content, specific activity, and subunit composition, confirm the usefulness of this method for obtaining highly active preparations of the enzymes.

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Identification of the mitochondrially encoded subunit 6 of F<inf>1</inf>F<inf>O</inf> ATPase in Trypanosoma brucei
2015, Molecular and Biochemical Parasitology
Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F₁F_O ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the ³⁵S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence.
Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W<inf>gm</inf>ZOflL
2015, FEBS Letters
Citation Excerpt :
The inhibitors were titrated to find out the lowest effective concentration. Complex III was isolated from dark-grown E. gracilis strain Z by anion-exchange chromatography as described in [24] Chromatographic protein fractions with the highest enzymatic activities of complex III were mixed together, concentrated by centrifugation through an Amicon XM100 ultrafilter and further characterized. The enzymatic activity of complex III was measured using various concentrations of cytochrome c (5–100 μM).
The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain W_gmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena.
Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei
2014, Molecular and Biochemical Parasitology
Citation Excerpt :
General 2D patterns of both complexes remain constant and small but notable differences in their relative migration probably reflect differences in the presence/absence of some subunits not connected with their activity. Indeed, the C. fasciculata and L. tarentolae complexes IV have 10 [53] and 11 [21] subunits, respectively, while in T. brucei, even more subunits have been characterized [31,50,51]. The in-gel staining of complex III previously described elsewhere [38,57] was non-specific in our hands.
Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites
Phosphatidylethanolamine and cardiolipin differentially affect the stability of mitochondrial respiratory chain supercomplexes
2012, Journal of Molecular Biology
The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.
Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits
2012, Molecular and Biochemical Parasitology
The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.
Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form
2011, Molecular and Cellular Proteomics
Citation Excerpt :
Nevertheless, this possible association does not appear to be essential for the structural integrity of either complex because repression of components of either complex that result in the loss of one complex do not result in the loss of the other complex (9). Purified complexes III from Crithidia fasciculate (13, 33) and L. tarentolae (14) consist of ten and eleven subunits, respectively. Whereas only six subunits were found in the complex purified from T. brucei.
The mitochondrial respiratory chain is comprised of four different protein complexes (I–IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F_oF₁-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.

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Experimental Parasitology

Abstract

References (20)

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

Biochimica et Biophysica Acta

Ubiquinol-cytochrome c oxidoreductase of higher plants: Isolation and characterization of the bc₁ complex from potato tuber mitochondria

Journal of Biological Chemistry

Detection of the mitochondrially encoded cytochrome c oxidase subunit I in the trypanosomatid protozoan Leishmania tarentolae: Evidence for translation of unedited mRNA in the kinetoplast

Journal of Biological Chemistry

Partial kinetoplast-mitochondrial gene organization and expression in the respiratory deficient plant trypanosomatid Phytomonas serpens

Molecular and Biochemical Parasitology

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c₁

Journal of Biological Chemistry

The trypanosomatid Rieske iron–sulfur proteins have a cleaved presequence that may direct mitochondrial import

Biochimica et Biophysica Acta

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis

Analytical Biochemistry

Characterization of a protein fraction containing cytochromes b and c₁ from mitochondria of Leishmania tarentolae

Experimental Parasitology

Characterization of the respiratory chain from cultured Crithidia fasciculata

Molecular and Biochemical Parasitology

Cited by (20)

Identification of the mitochondrially encoded subunit 6 of F<inf>1</inf>F<inf>O</inf> ATPase in Trypanosoma brucei

Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W<inf>gm</inf>ZOflL

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei

Phosphatidylethanolamine and cardiolipin differentially affect the stability of mitochondrial respiratory chain supercomplexes

Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits

Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form

Regular ArticleLeishmania tarentolae: A Parallel Isolation of Cytochrome bc1 and Cytochrome c Oxidase

Abstract

Biochimica et Biophysica Acta

Journal of Biological Chemistry

Journal of Biological Chemistry

Molecular and Biochemical Parasitology

Journal of Biological Chemistry

Biochimica et Biophysica Acta

Analytical Biochemistry

Experimental Parasitology

Molecular and Biochemical Parasitology

Regular Article
Leishmania tarentolae: A Parallel Isolation of Cytochrome bc₁ and Cytochrome c Oxidase