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PubMed1993

A transcription map of the chloroplast genome from rice (Oryza sativa).

The complete nucleotide sequence (134,525 bp) of the chloroplast genome from rice (Oryza sativa L.) contains four rRNA genes, 30 tRNA genes, and over 100 genes that encode proteins. However, expression of only a few of these genes, namely psbA, rbcL, and atpB/E, has been detected. We constructed the complete transcription map of rice chloroplast DNA by Northern hybridization of total RNA from rice seedlings, using subclones from a clone bank of rice chloroplast DNA as probes. Approximately 90% of the chloroplast genome was transcribed, as detected by a non-radioactive hybridization system. Most of the genes on the chloroplast DNA are organized as clusters and are co-transcribed as long primary transcripts. We identified 16 polycistronic transcripts from the rice chloroplast genome. Furthermore, the genes for components of photosystems I and II, the gene for the large subunit of RuBisCO, the genes for ATPase, the genes for components of the cytochrome complex, and the rRNA genes were expressed at the highest levels.

ATP Synthetase Complexes↗
PubMed1985

Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene-[35S]sulfonate.

Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein.

Animals↗
PubMed1980

Inhibition of electron transfer in the cytochrome b-c, segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone.

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochrome c reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochrome c reductase and ubiquinol-cytochrome c reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochrome c reductase activities is markedly dependent on pH in the range pH 7-8. At pH 7.0 inhibition occurs with an apparent Ki less than or equal to 1 x 10(-8) M, while at pH 8.0 the apparent Ki is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pKa at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in the b-c1 segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a function group in the b-c1 segment.

Animals↗
PubMed1984

Mitochondrial oxidative phosphorylation and respiratory chain: review.

Basic events concerning oxidative phosphorylation, i.e. the synthesis of ATP at the expense of respired oxygen at the level of mitochondria are described. Our knowledge concerning the functioning of respiratory chain, its structure, organization and topology inside the inner membrane of mitochondria has considerably improved in recent years. A central question--how does the respiratory chain cooperate with ATP-synthetase, also embedded in the inner membrane, to bring about the oxidative phosphorylation of ADP to ATP--has been one of the most challenging and difficult problems in biochemical research. The chemiosmotic hypothesis proposed by the British biochemist Peter Mitchell appears best in describing the basic events of the recovery of the redox energy liberated along the respiratory chain to synthesize ATP through a membrane process. Moreover the chemiosmotic hypothesis is not restricted to mitochondrial oxidative phosphorylation but appears to provide a general explanation to the synthesis of ATP in all transducing membranes: inner mitochondrial membrane, bacterial plasma membrane, thylakoid membranes in chloroplasts of green plants.

ATP Synthetase Complexes↗
PubMed1982

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis.

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively. In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).

Animals↗
PubMed1983

Effects of N, N'-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria.

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstituted mitochondrial cytochrome b-c1 complex. DCCD inhibits equally electron flow and proton translocation (i.e., the H +/- ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzyme activity and the cross-linking, suggesting that the two phenomena may be couples. Binding of [14C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.

Animals↗
PubMed1985

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase.

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.

Animals↗
PubMed1988

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans.

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.

Animals↗
PubMed1986

Isolation and amino acid sequence of the 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase.

The 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase was isolated by a series of chromatographic steps involving dissociation of the complex by urea and guanidine. A clear distinction between the 9.5 kDa protein and the 9.2 kDa protein described earlier [(1982) J. Biochem. 91, 2077-2085] by SDS-PAGE was only achieved when the electrophoresis was performed according to Schägger et al. [(1985) FEBS Lett. 190, 89-94; (1986) Methods Enzymol. 126, 22] because in this gel system the apparent molecular mass of the 9.5 kDa protein is shifted to 11 kDa. The amino acid sequence was determined by solid-phase Edman degradation of the whole protein up to amino acid residue 80 and of the proteolytic cleavage fragments. The protein consists of 81 amino acid residues; its Mr was calculated to be 9507. Structure predictions have been made from average and sided hydropathy profiles. The 9.5 kDa protein is either bound to the core proteins within a 9.5 kDa-core protein subcomplex or else it aggregates easily with the core proteins during the isolation procedure.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of the MHC linked beta-type proteasome subunit MC13 cDNA.

We have cloned and analysed the second mouse MHC-linked proteasome subunit, designated MC13, which appears to be homologous to the human RING10 proteasome protein. The isolated cDNA has an ORF encoding a protein of 276 amino acids with a molecular weight of ca. 30 kDa. Sequence alignment reveals that the subunit MC13 and several other mammalian proteasome subunits are encoded by a second proteasome gene family. This second gene family encodes subunits of the beta-type, reveals striking sequence similarities with the beta-subunit of archaebacterial proteasomes and is related to, but distinct from, the genes encoding the so-called alpha-type subunits.

Amino Acid Sequence↗
PubMed1995

Comparative study and cDNA cloning of the flavoprotein subunit of mitochondrial complex II (succinate-ubiquinone oxidoreductase: fumarate reductase) from the dog heartworm, Dirofilaria immitis.

Mitochondrial complex II functions as a fumarate reductase (FRD), the reverse reaction of succinate dehydrogenase (SDH), and plays an important role in the anaerobic respiratory chain of parasitic helminths. In this study, complex II from the dog heartworm, Dirofilaria immitis adult, which is thought to act as a homolactatic fermenter, was examined in terms of its enzymatic features and primary structure in order to investigate the possible role of mitochondria in this filaria. Mitochondria from D. immitis adult showed high FRD activity when the enzymatic assay was performed using methylviologen as an artificial electron donor. The ratio of SDH to FRD in D. immitis was comparable to that in Ascaris suum adult, which is known to have an anaerobic mitochondrial respiratory chain with a high FRD activity of complex II. The FRD activity of D. immitis mitochondria was inhibited by the sulfhydryl reagent N-ethylmaleimide (NEM), while that of A. suum complex II was resistant to this inhibitor. The presence of the flavoprotein (Fp) subunit, which contains the substrate binding active site, was confirmed in D. immitis mitochondria by immunoblotting using a monoclonal antibody against the A. suum Fp subunit. By homology probing with the polymerase chain reaction, the entire cDNA for the D. immitis adult Fp was cloned and sequenced. The deduced amino acid sequence showed significant homology to that of A. suum and other mitochondrial Fps, in contrast to much less similarity to bacterial FRD, even though the D. immitis complex II showed high FRD activity. These results are the first indication of the presence of a functional complex II in D. immitis mitochondria.

Amino Acid Sequence↗
PubMed1983

Isolation, characterization and regulation of expression of the nuclear genes for the core II and Rieske iron-sulphur proteins of the yeast ubiquinol-cytochrome c reductase.

Cloning and mapping of the yeast nuclear genes for the core II (Mr 40 000) and Rieske iron-sulphur proteins of the mitochondrial ubiquinol-cytochrome c reductase, and comparison with the genomic regions in nuclear DNA from which they are derived, show that the genes are likely to be present in single copies and that they are not closely linked. They have been reintroduced into yeast cells on multi-copy plasmids and, similar to results obtained for the Mr 11 000 subunit [Van Loon et al., EMBO J. 2 (1983) 1765-1770], increase in the dosage of either gene prompts discoordinate synthesis of the encoded protein. Quantitative analysis of transformants carrying extra copies of the gene for the iron-sulphur protein shows that messenger RNA level, rate of synthesis and steady-state concentration of the protein correlate well with each other. This indicates that its level, in contrast to that of the Mr 11 000 subunit, is only determined by the concentration of its messenger RNA. Over-production of these proteins does not interfere with mitochondrial function as judged from growth rates of transformed cells on non-fermentable media. The excess Mr 40 000 protein is imported into the mitochondrion, showing that import of this subunit is not obligatorily coupled to complex assembly.

Cloning, Molecular↗
PubMed2006

The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1.

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.

Animals↗