Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.9.3.1 (cytochrome oxidase)
8,822 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [3H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of D-glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-beta-D-glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni(2+)-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.
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PMID:Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae. 792 Jul 12

Functional plasma membranes from the filamentous fungus Penicillium chrysogenum have been isolated with the objective of studying transport processes. The isolation procedure consists of three steps, namely homogenization of cells with a Braun MSK homogenizer, followed by Percoll gradient centrifugation and floatation of membranes in a three-step Nycodenz gradient. This method can be applied to strains which differ significantly in morphology and penicillin-production capacity. Plasma membranes were fused with liposomes containing the beef heart mitochondrial cytochrome-c oxidase. In the presence of reduced cytochrome c, the hybrid membranes maintained a high proton motive force that functions as a driving force for the uptake of the amino acids arginine and valine via distinct transport systems.
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PMID:Structural and functional properties of plasma membranes from the filamentous fungus Penicillium chrysogenum. 792 75

An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.
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PMID:The HUP1 gene product of Chlorella kessleri: H+/glucose symport studied in vitro. 807 29

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A-->G3260 transition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho degrees). The presence and amount of the mutant G3260 vs the wild-type A3260 were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I- and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A-->G3260 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations.
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PMID:Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy. 813 49

We have performed experiments which demonstrate that puromycin inhibits the import of proteins into mitochondria in in vitro reactions containing mitochondria isolated from the yeast Saccharomyces cerevisiae and precursor proteins synthesized in a nuclease-treated rabbit reticulocyte lysate. Puromycin inhibited the import of several precursor proteins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III. The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin. Puromycin-induced import inhibition could be overcome by adding additional ATP to the import reactions. However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase with carboxyatractyloside, ATP addition was unable to overcome the inhibitory effect of puromycin on protein import. Collectively, these results demonstrate that puromycin inhibits protein import into mitochondria by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside the inner mitochondrial membrane. In addition to supporting the view that ATP is required in the matrix for efficient protein import, these results may provide a useful tool for identifying the ATP-binding components of the import apparatus.
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PMID:Puromycin inhibits protein import into mitochondria by interfering with an intramitochondrial ATP-dependent reaction. 833 41

We have utilized a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae, in addition to performing a series of in vivo experiments in yeast, to investigate the coupling between cytosolic protein synthesis and protein transport into mitochondria. We found that the import of bulk mitochondrial proteins was inhibited in both the homologous in vitro reaction and in vivo upon arrest of cytosolic protein synthesis with the addition of cycloheximide. Tight coupling of synthesis and import was also demonstrated in vivo for the beta subunit of the mitochondrial F1-ATPase. We also investigated the effect of the antifolate methotrexate on the import of a fusion protein consisting of the mitochondrial targeting signal of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (the COXIV-DHFR fusion protein). Methotrexate has previously been shown to inhibit posttranslational import of COXIV-DHFR by preventing the DHFR moiety from unfolding. However, we found that antifolate addition had no inhibitory effect on the import of COXIV-DHFR in vivo, suggesting that its import into mitochondria in yeast cells occurs cotranslationally. Further, when we treated yeast with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone to collapse the mitochondrial membrane potential and induce the accumulation of extramitochondrial precursor pools, we found that the ability to be imported by a strictly posttranslational mechanism upon reestablishing the membrane potential varied from one precursor to another, suggesting that cotranslational import may be mandatory for the import of some proteins in vivo. In summary, our findings are entirely consistent with the notion that import of proteins into yeast mitochondria occurs cotranslationally under normal conditions in vivo.
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PMID:Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo. 838 May 82

C3H mice were infected with 30 metacercarial cysts of either echinostome to study the pathological, ultrastructural, and cytochemical effects of the infection on the mouse small intestine. In mice infected with Echinostoma caproni, the intestine showed villous atrophy with fused or eroded villi. The microvilli of the enterocytes were sparse and distorted and showed reduced alkaline phosphatase activity. The crypts of Lieberkuhn were hyperplastic and showed a marked reduction in goblet and Paneth cells. As compared with uninfected controls, there was a marked reduction in glucose-6-phosphatase activity in the enterocytes of the infected gut. Collagen fibers and the number of fibroblasts were increased under the epithelium. In mice infected with E. trivolvis, the tips of the intestinal villi were bent and blunted. The microvilli of the enterocytes were less tightly packed than those of uninfected controls. The mitochondria in the enterocytes were irregularly shaped, contained intracristal bodies, and showed increased cytochrome oxidase activity as compared with those of uninfected controls. The crypts were hyperplastic but showed an increase in the numbers of goblet and Paneth cells. The fibroblasts and collagen fibers showed abnormal development. The ultrastructural and cytochemical differences seen in this study reflect the uniqueness of the host-parasite relationship of each of these echinostome species in the gut of the C3H mouse.
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PMID:Expulsion of Echinostoma trivolvis (Cort, 1914) Kanev, 1985 and retention of E. caproni Richard, 1964 (Trematoda: Echinostomatidae) in C3H mice: pathological, ultrastructural, and cytochemical effects on the host intestine. 839 78

It is still controversial whether cyanobacteria (blue-green algae) contain an aa3-type cytochrome-c oxidase. We have approached this problem using DNA analysis. Using a DNA probe coding for the most conserved part of subunit I of the Bacillus enzymes, structural genes for the oxidase of a thermophilic cyanobacterium Synechococcus vulcanus were cloned and sequenced. We found genes for subunits II, I, III and IV of this order like those of the Bacillus enzymes, and a terminator structure after the gene for subunit IV. The deduced protein sequences for the subunits II, I and III showed consensus amino-acid residues atevery important portion, suggesting that these genes are operating. However, the S. vulcanus oxidase lacked a cytochrome-c-moiety fused to subunit II, the 13th and 14th hydrophobic segments of subunit I which are lacking in the Paracoccus enzyme, and the 1st and 2nd ones of subunit III which are lacking in the Bacillus enzyme, were not found. A gene homologous to ctaB gene, which locates at the 5'-upstream region of the gene for subunit II and co-transcribed in Bacillus subtilis, was not found. Comparison of protein sequences showed that S. vulcanus cytochrome oxidase is closer to Bacillus cytochrome oxidases than the mitochondrial and Paracoccus enzymes, or quinol oxidases from B. subtilis and Escherichia coli.
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PMID:The genes in the thermophilic cyanobacterium Synechococcus vulcanus encoding cytochrome-c oxidase. 839 73

The disposition of the facial vibrissae of the mouse is represented as a matrix-like array of cell aggregates in rows and columns at every station of the whisker-to-barrel pathway. In order to evaluate the role of each station in this pathway, lesions were made in the facial vibrissae of the mystacial group on P0-P3, and the animals were sacrificed on P8. The effects of the lesions on the cell aggregates in the array were analyzed by using cytochrome oxidase and gallocyanin cell-staining methods. Division of cell aggregates in the array was controlled by row basis interactions through the pathway up to the cerebral cortex. In this organization, affected cell aggregates which corresponded to the damaged vibrissae were eliminated and/or fused together in the array of the thalamic relay nucleus. On the basis of thalamic modification, the final array of cell aggregates was remodelled in the cerebral cortex. In contrast, affected cell aggregates remained degenerative spaces at the original sites in the array in relation to the damaged vibrissae in the brain stem trigeminal nuclear complex. These results indicate that a protoframework with row basis orientation for the division of cell aggregates is prepared in every station of the pathway at the time of lesioning, and adjustment of subcortical alterations in the thalamic relay nucleus is a decisive process to let the cerebral cortex remodel the topographic array of cell aggregates.
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PMID:Remodelling in the array of cell aggregates in somatotopic representation of the facial vibrissae through the trigeminal sensory system of the mouse. 860 80

Systemic cytochrome oxidase deficiency presenting as Leigh syndrome is a well-defined biochemical entity. Although the enzyme defect is demonstrable in all tissues, clinical abnormalities are restricted to the central nervous system. Biochemical studies comparing rates of synthesis of cytochrome oxidase subunits with the steady-state levels of immunoreactive protein in the mitochondrial inner membrane suggest a defect in assembly or stability of the complex. Family studies suggest that the disease is inherited as an autosomal recessive and somatic cell genetic studies directly implicate nuclear genes. As there are likely to be a number of different nuclear genes involved in the synthesis, assembly and stability of the cytochrome oxidase complex, we have fused patient fibroblasts and analysed the heterokaryons for complementation of the enzyme defect in an attempt to define the extent of genetic heterogeneity in this condition. So far, three complementation groups have been defined, although the majority of patients fall into a single group.
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PMID:Complementation analysis of systemic cytochrome oxidase deficiency presenting as Leigh syndrome. 898 48


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