Biomedical subjects

D González-Halphen

Publications and source records attributed to D González-Halphen.

17 recordsLinked to original sources

PubMed2003

Structure, organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii.

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.

Amino Acid Sequence↗

PubMed2002

A case of Kearns-Sayre syndrome with the 4,977-bp common deletion associated with a novel 7,704-bp deletion.

Mitochondria from a patient diagnosed with Kearns-Sayre syndrome (KSS) exhibited severely diminished cytochrome c oxidase activity and at least four mitochondrial DNA (mtDNA) species: 9%-11% of the fulllength mtDNA (16.6 kb), 70%-75% of a 11.7-kb population (harboring the 4,977-bp common deletion), 2%-3% of a 10.5-kb population, and 12%-17% of a 8.9-kb population. The 8.9-kb mtDNA exhibited a secondary deletion that extended 7,704 bp from nucleotide 7,979 in the cox2 gene to nucleotide 15,683 in the cytb gene. To our knowledge, this is the first description of the presence of at least two large-scale deletions of mtDNA in KSS.

Blotting, Southern↗

PubMed2000

Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes.

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.

Amino Acid Sequence↗

PubMed2000

Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae.

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.

Amino Acid Sequence↗

PubMed2000

Polytomella spp. growth on ethanol. Extracellular pH affects the accumulation of mitochondrial cytochrome c550.

A defined medium with ethanol as sole carbon source was devised for growth of the colorless, unicellular alga Polytomella spp. Cell density on this carbon source was related to extracellular pH. An acidic pH was required for ethanol utilization; best yields were obtained at pH 3.7. Spectroscopic analysis of the cells showed that the concentration of cytochrome c per cell was 40% higher than at pH 6.0; the concentrations of cytochrome a606 (cytochrome c oxidase) and b566 (cytochrome bc1 complex) were the same. A soluble cytochrome c550 was purified from cells grown at pH 3.7 and characterized by peptide sequencing as the 12-kDa cytochrome c550 of the mitochondrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c550 at pH 3.7 than at pH 6.0. RNA blot analysis gave clear evidence of the abundance of c550 transcript in cells grown at pH 3.7. The amount of mitochondrial proteins obtained from cells grown at pH 3.7 was twofold higher than that of cells grown at pH 6.0. Mitochondria isolated from both cell types readily oxidized succinate, malate or ethanol. The rates of oxygen uptake were 20-25% higher in mitochondria from cells grown at pH 3.7. Cyanide and antimycin A inhibited respiration with succinate up to 95% in both types of mitochondria. The participation of cytochrome c550 in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.

Amino Acid Sequence↗

PubMed1998

Two unusual amino acid substitutions in cytochrome b of the colorless alga Polytomella spp.: correlation with the atypical spectral properties of the bH heme.

The dithionite-reduced spectra of the purified bc1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc1 complex from C. reinhardtii showed a profile similar to those of the bc1 complexes from other species. In contrast, the bc1 complex from Polytomella spp. exhibits a double-peak spectrum in the alpha-band region, where the absorption bands of cytochrome c1 and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b of Polytomella spp. was cloned, sequenced, and compared with that of C. reinhardtii. The Polytomella spp. cytochrome b gene is 1113 bp long and does not contain introns. The deduced protein sequence exhibits 56% identity and 68% similarity with the cytochrome b of C. reinhardtii, and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes of C. reinhardtii and C. smithii. A comparison of the primary sequences of the Polytomella spp. cytochrome b with other b-type cytochromes, and its analysis based on the structure featuring eight transmembrane stretches, allowed the identification of a tyrosine in position 114, which substitutes for a tryptophan present in all mitochondrial b-type cytochromes sequenced to date. In addition, the primary sequence of the cytochrome b from Polytomella spp. has a serine at position 36, instead of a nonpolar residue (alanine or leucine) found in all other species. In the proposed model for cytochrome b, both residues Tyr114 and Ser36 are in close proximity to the high-potential bH heme. The above data suggest that the polar residues Y114 and S36, each one by itself or in combination, may interact with heme bH of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.

Amino Acid Sequence↗

PubMed1998

An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum.

Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.

Animals↗

PubMed1997

Characterization of the alpha and beta-subunits of the F0F1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii.

The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl-beta-D-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases. The alpha-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G. and Franzén L.-G. (1996) Plant Mol. Biol. 31, 1105-1116). In whole mitochondria, the alpha-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature alpha-subunit. Subunit beta was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than alpha, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This beta-subunit is 7-8 kDa larger than the beta-subunits of other mitochondrial ATPases. It is suggested that the beta-subunit from Polytomella spp. may have a C-terminal extension similar to that described for the green alga C. reinhardtii (Franzén, L.-G. and Falk, G.(1992) Plant Mol. Biol. 19, 771-780). In addition, it was found that the C-terminal extension of the beta-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the epsilon-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the beta-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.

Amino Acid Sequence↗

PubMed1996

The deduced primary structure of subunit I from cytochrome c oxidase suggests that the genus Polytomella shares a common mitochondrial origin with Chlamydomonas.

We cloned and sequenced the mitochondrial gene encoding subunit I of cytochrome c oxidase (coxI) of Polytomella spp., a colorless alga related to Chlamydomonas. The purpose was to explore whether homology between the two species also exists at the level of a mitochondrial enzyme. The gene is 1512 bp long and contains no introns. The translated protein sequence exhibits 73.8% identity with its Chlamydomonas reinhardtii counterpart. The data obtained support the hypothesis that the separation of the colorless alga from the Chlamydomonas lineage was a late event in evolution, that occurred after the endosymbiotic process that gave rise to mitochondria.

Amino Acid Sequence↗

PubMed1995

A novel mitochondrial DNA point mutation associated with mitochondrial encephalocardiomyopathy.

A novel mitochondrial DNA (mtDNA) mutation at position nt 4320 in the tRNA(Ile) gene was associated with severe encephalopathy in a 7-month-old infant, who died of intractable hypertrophic cardiomyopathy. The mutation was present in heteroplasmic fashion (88%) in muscle and fulfills accepted criteria for pathogenicity. This is the fourth pathogenic mutation identified in this gene, which appears to be a "hotspot" for deleterious mutations affecting the heart. This report adds to the evidence of genetic heterogeneity in hypertrophic cardiomyopathies.

Base Sequence↗

PubMed1995

Characterization of a 5025 base pair mitochondrial DNA deletion in Kearns-Sayre syndrome.

We characterized a mitochondrial DNA deletion in a patient with Kearns-Sayre syndrome. Southern blot hybridization showed that 86 to 93% of the mitochondrial genome harbored a 5.0 kb deletion. The percentage of affected genomes is higher than in previously described cases. Direct sequencing of the breakpoint region revealed that the deletion extended 5025 bp from nt 10,050 in the tRNA Gly gene to nt 15,076 in the cytochrome b gene, thus 30% of the total mitochondrial genome was lost by this deletion. A pair of extremely short mirror sequences flanking the mitochondrial DNA breakpoints were identified. These flanking sequences differ from previously published consensus 'hot-spots', known to give rise to deletions in human mitochondrial DNA.

Adolescent↗

PubMed1995

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) is a novel inhibitor of electron transfer through the QN center of the mitochondrial bc1 complex.

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) inhibited the NADH- or succinate-supported rate of O2 consumption in beef heart submitochondrial particles (Ki = 7 x 10(-7) M). Oxygen comsumption was restored with the addition of ascorbate/TMPD, indicating that the inhibitory effect was on the ubiquinol-cytochrome c reductase activity of the respiratory chain. Difference spectra with submitochondrial particles indicated that DBHBM blocked electron transport through the cytochrome bc1 complex, in a mode closely similar to that of antimycin A. The reduction rates of cytochrome b by succinate were strongly inhibited in the presence of DBHBM plus myxothiazol, but not by DBHBM plus antimycin A. These data suggest that DBHBM may bind primarily to the QN center. In the purified bc1 complex, DBHBM and antimycin A induced a red shift from 562 to 566 nm of the alpha peak of cytochrome b, supporting the idea that DBHBM influences predominantly the ligand field of the b562 (bh) heme. Difference spectra in the presence or absence of myxothiazol showed that DBHBM induced the same red shift with a maximum at 565 nm and a minimum at 559 nm. We conclude that DBHBM blocks electron transfer at the QN center and thus may be considered a novel group III inhibitor of the bc1 complex.

Animals↗

PubMed1995

Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide.

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

Amino Acid Sequence↗

PubMed1994

A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.

The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.

Amino Acid Sequence↗

PubMed1993

Subunit structures of purified beef mitochondrial cytochrome bc1 complex from liver and heart.

The existence of tissue-specific isozymes of cytochrome c oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrial bc1 complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochrome c oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels of bc1 complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochrome c oxidase, bc1 complexes from liver and heart do not exhibit tissue-specific differences.

Amino Acid Sequence↗

PubMed1991

On the interaction of mitochondrial complex III with the Rieske iron-sulfur protein (subunit V).

Limited proteolysis of solubilized beef heart mitochondrial complex III with trypsin yields a product previously identified as fragment V" (González-Halphen, D., Lindorfer, M. A., and Capaldi, R. A. (1988) Biochemistry 27, 7021-7031). In this work, fragment V" was generated by trypsin treatment of both the intact complex III and the purified Rieske iron-sulfur protein. Thus, in its bound or isolated form, the same sites of subunit V are sensitive to protease action. Fragment V" was a soluble protein that retained its iron-sulfur moiety. It was purified by exclusion from a hydrophobic phenyl-Sepharose CL-4B column followed by gel filtration. In contrast to the pure, intact subunit V, fragment V" did not reconstitute oxidoreductase activity when combined with complex III devoid of subunit V. However, a 20-amino acid synthetic peptide carrying the sequence between amino acids Lys33 and Lys52 of the Rieske iron-sulfur protein competed with intact subunit V in reconstitution assays. The results obtained suggest that the iron-sulfur protein binds to complex III by hydrophobic protein-protein interactions, and that a nontransmembrane 18-amino acid amphipathic stretch accounts for the association of this subunit to the rest of the complex.

Amino Acid Sequence↗

PubMed1988

Subunit arrangement in beef heart complex III.

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Schägger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.

Amino Acid Sequence↗