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PubMed1989

Inhibitory effects of tetragalloylglucose on the complex II of mitochondrial respiratory chain of Ascaris muscle.

The effects of tetragalloylglucose (1,2,3,6-tetra-O-galloyl-beta-D-glucose) on purified complex II (succinate-ubiquinone oxidoreductase) of the mitochondrial electron transport system of Ascaris muscle were studied. Both succinate-ubiquinone-1 (Q1) oxidoreductase, and succinate dehydrogenase measured with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) in the presence of phenazine methosulfate (PMS) were inhibited by tetragalloylglucose. The inhibitions of both reductase activities of complex II were of competitive type, and the inhibitor constant (Ki) for Ascaris complex II (148 nM) was lower than that for rat liver complex II (1.5 microM). Thus, Ascaris complex II is much more sensitive to this inhibitor than the mammalian counterpart.

Animals↗
PubMed2001

Accessory proteins that control the assembly of MHC molecules with peptides.

The stable assembly of Major Histocompatibility Complex (MHC) molecules with peptides is controlled by a number of cofactors, including proteins with general housekeeping functions and proteins with dedicated functions in MHC assembly. Recent work in my laboratory has focused on two chaperones, tapasin (tpn) and DM, that play critical roles in the loading of peptides onto MHC class I and MHC class II molecules, respectively. Tapasin is a transmembrane protein that tethers empty class I molecules in the endoplasmic reticulum to the transporter associated with antigen processing. DM is a peptide exchange factor that binds with empty and peptide-loaded class II molecules in endosomal and lysosomal compartments. Although a number of different functions for tapasin and DM have been proposed, emerging evidence suggests that both of these chaperones retain unstable MHC molecules in peptide-loading compartments until they bind with high-affinity peptides. These cofactors therefore promote the surface expression of long-lived MHC-peptide complexes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

Isolation of complex III from various mitochondria. Comparison of the structural and functional properties of the preparations from beef heart, calf liver and Neurospora crassa.

Active complex III was isolated by an improved procedure from beef heart mitochondria, from Neurospora crassa mitochondria and for the first time from mitochondria originating from mammalian tissue other than heart, i.e. calf liver. The described procedure consists of differential extraction of the respective mitochondria, hydroxyapatite chromatography and, finally, either gel- or affinity chromatography. The preparations contain the well known prosthetic groups, i.e. 6-8 mumol b-type heme, 3-4 mumol c-type heme and 5-8 mumol non-heme iron per g of protein. The preparations from beef heart and from calf liver mitochondria are indistinguishable in their subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis, whereas the preparation from Neurospora crassa mitochondria is clearly different. The phospholipid content of all preparations is rather low, amounting to about 100 mumol/g protein. The molar catalytic activity of ubiquinol-9-cytochrome c reductase at 25 degrees C amounts to 50s-1 for the N. crassa complex III and 70-100s-1 for the bovine complexes. After reincorporation into phospholipid vesicles, all preparations how tight coupling between electron transfer from ubiquinol to cytochrome c and proton translocation across the phospholipid bilayer.

Animals↗
PubMed1987

Identification and properties of a quinol oxidase super-complex composed of a bc1 complex and cytochrome oxidase in the thermophilic bacterium PS3.

Evidence for the presence of a quinol oxidase super-complex composed of a cytochrome bc1 complex and cytochrome oxidase in the respiratory chain of a Gram-positive thermophilic bacterium PS3 is reported. On incubation with an octyl glucoside-solubilized fraction of the total membranes of PS3 anti-serum against PS3 cytochrome oxidase gave an immunoprecipitate that showed both quinol-cytochrome c reductase and cytochrome c oxidase activities. When the cholate-deoxycholate and LiCl-treated membranes of PS3 were solubilized and subjected to ion-exchange chromatography in the presence of octaethyleneglycol dodecyl ether, most of the A-, B-, and C-type cytochromes were copurified as a peak having both quinol-cytochrome c reductase and cytochrome oxidase activities. The immunoprecipitate and quinol oxidase preparation contained hemes a, b, and c in a ratio of about 2:2:3, indicating the presence of one-to-one complex of cytochrome oxidase containing 2 hemes a and one heme c, and a bc1 complex containing 2 hemes b and 2 hemes c. Gel electrophoresis in the presence of dodecyl sulfate showed that the immunoprecipitate and quinol oxidase preparation were composed of seven subunits; those of 51 (56-kDa), 38, and 22 kDa for cytochrome oxidase and those of 29, 23, 21, and 14 kDa for the bc1 complex. The 38-, 29-, and 21 kDa components possessed covalently bound heme c. The apparent molecular mass of the super complex was estimated to be as 380 kDa by gel filtration.

Antibodies↗
PubMed1985

Electron paramagnetic resonance studies of mammalian succinate dehydrogenase. Detection of the tetranuclear cluster S2.

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.

Animals↗
PubMed1985

The essentiality of coenzyme Q for bioenergetics and clinical medicine.

Coenzyme Q is an essential component of the respiratory chain, where it represents a mobile pool between dehydrogenases and cytochromes. The fact that Q is a free component, and its concentration is not in great excess over the Km of the respiratory complexes, renders this compound potentially rate-limiting in the respiratory chain. On the other hand, the rate of lateral diffusion of Q in the mitochondrial membrane is not a limiting step under physiological conditions. Quinoid compounds, which act as inhibitors of the respiratory chain at the level of Q, besides being useful tools for investigations of electron transfer, could be important in pathology as inhibitors of respiration.

Animals↗
PubMed1986

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans.

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.

Cell Membrane↗
PubMed1984

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative.

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.

ATP Synthetase Complexes↗
PubMedDate not supplied

[Effect of visible light on the ATP synthetase function of mitochondria].

Dependence of oxidative phosphorylation in rat liver mitochondria on redox state of endogenous flavoenzymes was studied. Regulation of the redox state of endogenous flavins in mitochondria was performed by visible light illumination of lambda = 380 nm in oxygen free as well as in oxygenated media. A decrease of oxidative phosphorylation was obtained at full reduction of flavins (respiratory control and ATP synthesis were measured); this effect was partially recovered when flavin was reoxidized by oxygen. Results of ESR measurements of the signal gl = 2.00 associated with semireduced flavins were in accordance with the appearance of diamagnetically completely reduced form of flavin. These facts may be considered as evidence of the regulative role of flavins in oxidative phosphorylation.

ATP Synthetase Complexes↗
PubMed1984

Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron transfer in rat liver mitochondria.

The primary effect of dicyclohexylcarbodiimide (DCCD) at the cytochrome b-c1 region of the respiratory chain of rat liver mitochondria is an inhibition of proton translocation. No significant decrease was observed in the rate of electron flow from succinate to cytochrome c when measured as cytochrome c reductase, K3Fe(CN)6 reductase, or the rate of H+ release in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone after treatment with sufficient DCCD to abolish completely electrogenic proton ejection. The inhibitory effects of DCCD were time and concentration dependent and affected by the pH of the medium. Lowering the pH from 7.3 to 6.7 resulted in a progressively faster rate and extent of inhibition of proton ejection by DCCD. At pH 6.9, the H+/2e- decreased by 50% within 30 s after DCCD addition; however, at pH 7.3, a 50% decrease was not observed until 2 min after DCCD addition. DCCD did not act as an uncoupler as both the rate of proton ejection and back decay were decreased after incubation with DCCD. Treatment of rat liver mitochondria with DCCD under these same conditions also resulted in a broadening of the sharp spectral shift of cytochrome b observed after antimycin addition to mitochondria previously reduced with succinate suggesting that DCCD may modify cytochrome b in such a way that the binding of antimycin is altered.

Animals↗
PubMed1983

Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue.

Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.

ATP Synthetase Complexes↗
PubMed1983

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase.

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.

ATP Synthetase Complexes↗
PubMedDate not supplied

[Effect of specific inhibitors of the enzymes of the respiratory chain and ATP-synthetase on ion transport in mitochondria induced by non-enzymic peroxide reactions].

Effect of specific inhibitors of membrane proton pumps of the respiratory chain and ATP-synthetase on ionic permeability of mitochondrial membranes induced by bivalent ferrum ions or organic hydroperoxide was studied. A conclusion is made concerning the existence of functional relations between non-specific oxidative reactions of the membranes with membrane proton pumps on the one hand and transport potassium ions in mitochondria on the other.

ATP Synthetase Complexes↗