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At least 1,099 records · Page 61Linked to original sources
PubMed1986

Triphasic reduction of cytochrome b and the protonmotive Q cycle pathway of electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain.

Reduction of cytochrome b in isolated succinate-cytochrome c reductase is a triphasic reaction. Initially, there is a relatively rapid, partial reduction of the cytochrome b, the rate of which matches the rate of reduction of cytochrome c1. This is followed by partial or complete reoxidation of the b, which is then followed by slow rereduction. At very low concentrations of succinate, the initial partial reduction of b is followed by reoxidation, but the third (rereduction) phase is absent, owing to insufficient substrate to rereduce the cytochromes. If antimycin is added at various times during the triphasic reaction, it inhibits the reoxidation and also inhibits the rereduction phase. Antimycin does not inhibit the initial phase of b reduction and, if added before or during this phase, it causes reduction of b to proceed to completion as a monophasic reaction. Myxothiazol inhibits the first phase of b reduction and the subsequent reoxidation, but does not inhibit the third, slow phase of b reduction. The resulting monophasic reduction of b which is observed in the presence of myxothiazol is slower than that in the presence of antimycin. The combination of both inhibitors, whether added together or successively during the triphasic reaction, completely inhibits b reduction. The triphasic reduction of cytochrome b is consistent with electron transfer by a protonmotive Q cycle in which there are two pathways for cytochrome b reduction. One pathway allows the initial phase of cytochrome b reduction by a myxothiazol-sensitive reaction in which reduction of b by ubisemiquinone is linked to reduction of iron-sulfur protein and cytochrome c1 by ubiquinol. In the second phase of the triphasic reaction, the b cytochromes are reoxidized by ubiquinone or ubisemiquinone through an antimycin-sensitive reaction. If oxidation of ubiquinol by iron-sulfur protein is blocked, either by myxothiazol or by reduction of iron-sulfur protein and cytochrome c1, the b cytochromes can be reduced by reversal of the antimycin-sensitive pathway, thus accounting for the third phase of b reduction.

Antimycin A↗
PubMed1985

Chicken heart ubiquinol: cytochrome c reductase--isolation by affinity chromatography and resolution of enzyme into subunits.

Chicken heart mitochondrial ubiquinol: cytochrome c reductase has been isolated as a monodisperse form by solubilization with Triton X-100 in low ionic strength and subsequent affinity chromatography. The enzyme complex contains less than 0.15 mumol ubiquinone and 12 mumol phospholipid per mumol enzyme. The spectrum of cytochrome b in the reduced exhibited two maximum alpha-absorption bands at 558 nm and 562 nm; a shoulder at 553 nm for c1. The purified enzyme complex consists of at least 9 subunits with apparent molecular weight of 47 K, 44 K, 36 K, 31 K, 25 K, 17 K, 15 K, 12 K and 9 K. The enzyme complex has been dissociated into subdomains of subunits in the presence of increasing concentration of salt and Triton X-100. Three subunits carrying the redox centers (cytochrome b, cytochrome c1 and Fe-S protein) have been further isolated.

Animals↗
PubMed1986

[Succinate-ubiquinone reductase site of the respiratory chain].

Data on succinate-ubiquinone reductase are critically reviewed. The structural and catalytic properties of succinate dehydrogenase and succinate-ubiquinone reductase are compared. The redox components, active centers and proteins involved in the enzyme interaction with ubiquinone are described. Some structural and kinetic features of the succinate-ubiquinone reductase as the respiratory chain component and feasible mechanisms of regulation of the succinate-ubiquinone reductase activity are discussed.

Animals↗
PubMed1984

The preferential binding of dicyclohexylcarbodiimide to cytochrome b and phospholipids in soluble complex III from yeast mitochondria.

The binding of [14C]dicyclohexylcarbodiimide (DCCD) to soluble complex III from yeast mitochondria was examined under conditions which resulted in the inhibition of proton ejection but had a minimal effect on cytochrome c reductase activity. Incubation of the complex with 50-100 nmol of [14C]DCCD/nmol of cytochrome b at 12 degrees C did not result in any changes in the appearance of the high-molecular-weight subunits (I-V) after sodium dodecyl sulfate-gel electrophoresis, although a slight broadening of the three lowest molecular-weight subunits (VI-VIII) was observed. The [14C]DCCD was bound preferentially to subunit III (cytochrome b) and a wide band with an apparent low-molecular weight ranging from 8000 to 9000 to less than 2000 depending on the gel system used. Extraction of the [14C]DCCD-treated complex III with chloroform:methanol had no effect on subunit III but completely removed the low-molecular-weight radioactive band. Thin-layer chromatography of the chloroform:methanol extract revealed that the radioactive material extracted from the [14C]DCCD-treated complex III migrated with the same apparent RF as either free [14C]DCCD or cardiolipin. Amino acids were not detectable in an acid hydrolysate of the chloroform:methanol extract, suggesting the absence of protein. Digestion of the [14C]DCCD-treated complex III with either chymotrypsin or Staphylococcus aureus V8 protease resulted in the decrease of both staining intensity and labeling in subunit III but had no effect on the radioactivity in the low-molecular-weight material. These results confirm that DCCD binds preferentially to cytochrome b in complex III from yeast mitochondria and suggest that cytochrome b may play an important role in proton translocation at this site of the respiratory chain.

Binding Sites↗
PubMed1983

Non-random patterns of nucleotide substitutions and codon strategy in the mammalian mitochondrial genes coding for identified and unidentified reading frames.

The base sequence of large part of the mitochondrial DNA of Wistar rats is presented. The sequence is compared with those of other mammalian mitochondrial DNAs. The nucleotide and amino acid homologies, codon strategy, nature and patterns of substitutions are reported. It results a very high amount of silent substitutions and, in short divergence time, a predominance of transitions on transversions. In both types of substitutions a strong bias in avoiding the use of the G in the third codon position is observed.

ATP Synthetase Complexes↗
PubMed1980

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c.

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.

Animals↗
PubMed1995

Association of LMP2 and LMP7 genes within the major histocompatibility complex with insulin-dependent diabetes mellitus: population and family studies.

LMP2 and LMP7, two subunits of the proteasomes encoded in the major histocompatibility complex, are speculated to play a role in the generation of endogenous peptides for presentation by class I molecules to cytotoxic T cells. Their possible role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) has not been documented. In this study of Caucasian subjects, we have analyzed the polymorphisms of four genes within the HLA class II region (LMP2, LMP7, and HLA-DRB1 and -DQB1) in 198 unrelated IDDM patients and 192 normal controls ascertained from the southeastern United States. A genomic polymorphism of LMP7 was found strongly associated with IDDM, and the Arg/His-60 polymorphism in LMP2 was found associated with IDDM only in subjects containing an HLA DR4-DQB1*0302 haplotype. To determine whether the apparent associations between LMP genes and IDDM resulted from the strong linkage disequilibria observed between LMP and HLA-DR/DQ genes, we compared LMP gene frequencies in extended LMP-HLA haplotypes derived from control and diabetic families. Our results suggest that LMP genes have independent effects on IDDM susceptibility.

Autoimmune Diseases↗
PubMed1993

PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins.

We have cloned the yeast PRE2 gene by complementation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a beta-type member of the proteasomal gene family, is essential for life and codes for a 287-amino acid proteasomal subunit with a predicted molecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to enhanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human Ring10 protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class II region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to Ring10 implies the hypothesis that Ring10 is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might further suggest that replacement of constitutive proteasomal components by functionally related major histocompatibility complex-linked low molecular mass polypeptides, as is Ring10, adapts mammalian proteasomes for functions in the immune response.

Amino Acid Sequence↗
PubMed1997

Evolution of proteasome subunits delta and LMP2: complementary DNA cloning and linkage analysis with MHC in lower vertebrates.

The class II region of the mammalian MHC harbors two proteasome subunit genes, LMP2 and LMP7. These genes are induced by IFN-gamma, and their products are incorporated into proteasomes substituting for their closest relatives, the delta and X subunits, respectively. This substitution is believed to change the proteolytic specificity of proteasomes, making it more suitable for generation of peptides to be presented by class I molecules. To elucidate the phylogenetic origin of LMP2 and the linkage of its gene with the MHC, reverse transcriptase-PCR amplification of Xenopus laevis and lamprey liver mRNA was performed with primers designed to amplify both the mammalian LMP2 and delta sequences. Both LMP2 and delta were amplified from X. laevis, whereas only delta was amplified from lamprey, suggesting that delta/LMP2 gene duplication occurred after divergence of cyclostomes but before divergence of amphibians. The linkage between the LMP2 gene and the MHC was observed in a diploid Xenopus species, Xenopus tropicalis, but not in a tetraploid species, X. laevis, indicating that this linkage was established before the divergence of amphibian from higher vertebrates, but that this linkage was lost in X. laevis, probably by a gene reorganization accompanying the tetraploidization. The X. laevis LMP2 and LMP7 mRNA showed a similar tissue distribution, indicating that the genetic linkage is not required for apparently coordinated tissue-specific expression of these genes. Sequence and linkage analyses suggest that LMP2 may not play as vital a role as LMP7 in Ag presentation.

Amino Acid Sequence↗
PubMedDate not supplied

[Redox state of the electron-transport carriers in cardiac mitochondria: a study by the method of low-temperature EPR spectroscopy].

The redox state of the carriers of electron-transport chain of cardiac mitochondria was studied in the conditions of normal perfusion, global ischemia and reoxygenation of the myocardial tissue. Experiments were performed on isolated rat hearts perfused at 37 degrees C by the "working heart" procedure. The EPR spectra of the freeze-clamped hearts were measured at 6-30 K. An analysis of the main values of g-tensor, line-shape, line-width and relaxation parameters of the components of low-temperature EPR spectra allowed to distinguish the signals from Fe-S centers of NADH-CoQ reductase and succinate-CoQ reductase, and the signals from free radical species of coenzyme Q and flavin coenzymes. The EPR spectra of hearts that were fixed during control perfusion and reperfusion contained predominantly the signal of oxidized S3 center of succinate-CoQ reductase. The free radical signal in these conditions was mainly due to ubisemiquinones. Besides the intensive signal of S3 center, the low-temperature EPR spectra contained also signals from different Fe-S centers paramagnetic in reduced state. The global ischemia of cardiac muscle caused essential reduction of the Fe-S clusters of the mitochondrial electron-transport chain. In ischemic condition the free radical EPR signal was mainly due to flavosemiquinones. The changes of the redox state of carriers of the mitochondrial respiratory chain correlated with the changes of the physiological parameters of cardiac muscle.

Animals↗
PubMedDate not supplied

The role of the proteasome in autoimmunity.

Type 1 diabetes is believed to be caused by T cell-mediated autoimmunity, with a prediabetic state characterized by the production of autoantibodies specific for proteins expressed by pancreatic beta cells. The non-obese diabetic (NOD) mouse is a spontaneous model of Type 1 diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) region of the genome. A specific proteasome defect has now been identified in NOD mouse lymphocytes that results from down-regulation of expression of the proteasome subunit LMP2, which is encoded by a gene in the MHC genomic region. This defect both prevents the proteolytic processing required for the production and activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which plays an important role in immune and inflammatory responses, in addition to increasing the susceptibility of the affected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). The proteasome dysfunction is both tissue- and developmental stage-specific and likely contributes to disease pathogenesis and tissue targeting.

Animals↗
PubMed1993

Genomic organization and tissue expression of mouse proteasome gene Lmp-2.

The mouse Lmp-2 gene is located within the major histocompatibility complex (MHC) class II region and encodes a subunit of the 20S cytosolic proteasome. Previous studies indicated that the 20S proteasome is a catalytic core of the 26S proteolytic complex that possesses a latent multicatalytic proteinase activity and catalyzes an ATP-dependent, selective breakdown of proteins ligated to ubiquitin. This complex has recently been postulated to be involved in the processing of endogenous antigenic peptides for the MHC class I pathway. Here, we report the genomic organization and tissue expression of the mouse Lmp-2 gene. We have cloned and sequenced the entire mouse Lmp-2 gene, including 5'- and 3'-flanking regions. The gene consists of six exons, and its genomic organization is very similar to that of the recently described human LMP2 gene. Putative promoter and enhancer elements were identified in the 5'-flanking region by sequence comparison with known consensus sequences. The Lmp-2 gene is expressed in most tissues of unstimulated mice, except for brain tissue. The comparison of the 5'-flanking region of human and mouse sequences is discussed.

3T3 Cells↗
PubMed1999

Involvement of oxidative stress on the impairment of energy metabolism induced by A beta peptides on PC12 cells: protection by antioxidants.

Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta) aggregates. In this study, the involvement of oxidative stress on A beta-induced energy metabolism dysfunction was evaluated on PC12 cells. It was shown that A beta peptides (A beta25-35 and A beta1-40) induce a concentration-dependent accumulation of reactive oxygen species (ROS), decrease the cellular redox activity, and lead to the depletion of ATP levels. The observed inhibition by A beta of mitochondrial function and of glycolysis is blocked by the antioxidants vitamin E, idebenone, and GSH ethyl ester. Taken together, these data suggest that exposure of PC12 cells to A beta results in an impairment of energy metabolism, leading to a deficit in ATP levels and to the compromise of cellular viability. Furthermore, the generation of ROS seems to be a crucial event responsible for the energetic metabolic dysfunction induced by A beta.

Adenosine Triphosphate↗