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PubMed1984

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2. [5-(Dimethylamino)-1-naphthoyl]adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier.

The binding to the ADP/ATP carrier in mitochondrial membranes of the 3'-O-(dimethylamino)naphthoyl (DAN) derivatives of AMP, ADP, and ATP was quantitatively analyzed. The sidedness of the fluorescent type binding to the "m" side only was shown comparing the mitochondrial membranes in various stages of integrity and surface orientation. In particles displacement by bongkrekate (BKA) is direct, whereas in the case of carboxyatractylate (CAT) the requirement for ADP and ATP demonstrates the transition from the "m" to the "c" state. Quantitatively the "physical" binding of [3H]DAN-AMP and fluorescence are well correlated, allowing for a little nonfluorescent binding to the c side. For DAN-AMP KD is 1.6 microM, for DAN-ADP KD is 0.8 microM, and in the Hill plot a straight line with n = 1.25 is obtained. The maximum number of binding sites for [3H]DAN-AMP (1.5 mumol/g of protein) is about equal to the sites found for [3H]BKA if the unspecific binding of both ligands is differentiated by blocking carrier sites with CAT. [3H]CAT binding is somewhat lower in accordance with the limited access of CAT to inverted vesicles. ADP is able to decrease fluorescence only by about 35% at high concentrations (10 mM) whereas GDP has virtually no effect. With ADP, DAN-AMP binding decreases by 30% of the total binding sensitive to BKA. Binding to ATPase is low because of the absence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1984

The reaction mechanism of the mitochondrial pyridine nucleotide transhydrogenase. A study utilizing arylazido-pyridine nucleotide analogues.

The addition of a purified mitochondrial pyridine nucleotide transhydrogenase enzyme preparation to complex I (NADH-CoQ reductase) results in a significant increase in the NADPH-AcPyAD+ transhydrogenase activity of the complex without influencing the NADH-AcPyAD+ transhydrogenase activity. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of complex I, the purified transhydrogenase enzyme preparation was found to co-migrate with the Mr = 130,000 (130K) subunit of the NADH-CoQ reductase. Loss of the NADPH-NAD+ transhydrogenase activity of complex I following limited tryptic digestion was associated with a corresponding loss of the 130K subunit from the complex. These results suggest that the 130K subunit of complex I is the specific peptide responsible for the catalysis of the NADPH-NAD+ transhydrogenase activity observed in complex I. Studies have been carried out testing the influence of photoaffinity pyridine nucleotide probes on the NADPH-NAD+ transhydrogenase activity catalyzed at three levels of resolution, i.e. a homogeneous transhydrogenase preparation, a partially resolved membrane preparation (complex I), and an intact mitochondrial membrane preparation (EDTA particles). Such studies have revealed arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+) to be a potent inhibitor and an active site-directed reagent for NADPH-NAD+ transhydrogenation at all three levels of resolution. On the other hand, arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)NAD+ does not produce a significant degree of inhibition of NADPH-NAD+ transhydrogenase activities prior to or following photoirradiation. Nevertheless, the NAD+ analogue has been found to specifically label, covalently, the transhydrogenase protein following photoirradiation of an enzyme-analogue mixture. Arylazido-beta-alanyl NAD+ can as well function as a substrate during transhydrogenation by virtue of being able to accept a hydride ion from NADPH. An interpretation of the observed nucleotide photoprobe specificity for interaction at the active site for transhydrogenation is advanced. In this interpretation, an ordered binding of substrate involves an initial NADP(H) (or NADP+ photoprobe) interaction with a hydrophobic region at the transhydrogenation site. This initial reactivity is followed by a positioning of NAD(H) (or the NAD+ photoprobe analogue) above or periphery to the NADP(H) nucleotide present at the active site region. Supportive evidence for this model for transhydrogenation is presented and discussed.

Affinity Labels↗
PubMed1990

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.

Animals↗
PubMed2002

2, 4-diamino-6- hydroxy pyrimidine inhibits NSAIDs induced nitrosyl-complex EPR signals and ulcer in rat jejunum.

BACKGROUND: It has been suggested that one aspect of non-steroidal anti-inflammatory drugs induced intestinal damage is due to either uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport. We investigated the latter possibility using electron paramagnetic resonance spectroscopy. RESULTS: Electron paramagnetic studies of NSAIDS on sub-mitochondrial particles revealed that indomethacin, but not with nabumetone, bound to a site near to Complex I and ubiquinone to generate a radical species. Normal rats exhibited prominent [3Fe-4S]ox signals (g approximately 2.01) at 20 K. One hour after indomethacin there was a prominent, intense and broad absorption pattern at (g approximately 2.07) suggesting, appearance of radical species overlapping [3Fe-4S]ox and was unaffected by pretreatment with 2,4 diamino -6-hydroxy pyrimidine. At 24 hrs, when macroscopic ulcers were seen, there was a new signal due to a nitric oxide radical (NO*). In contrast, nabumetone and 2,4 diamino-6-hydroxy pyrimidine pre-treated animals receiving indomethacin exhibited electron paramagnetic resonance spectra identical to those of controls at 24 hrs and neither was associated with small intestinal ulcers. Indomethacin and 2,4 diamino hydroxy pyrimidine pre-treated rats, but not nabumetone, had increased intestinal permeability. CONCLUSION: The results suggest that the in vivo effects of indomethacin modulate the mitochondrial respiratory chain directly at 1 h and 24 h through formation of nitric oxide. NO* appears to play an important role in the late pathogenic stages of NSAID enteropathy and may be the site for targeted treatment to reduce their toxicity.

Animals↗
PubMed1993

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1-ATPase beta subunit.

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1-induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron-transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity. IF1 antibodies have been used to show that IF1 can bind not only to the beta subunit of F1-ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339-1344] but also to a protein present in the inner-mitochondrial membrane. The cross-linking of IF1 to this membrane protein gave a product of M(r) 15000-16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross-linked product was obtained by using a cleavable cross-linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent M(r) 5000-6000. The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross-linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X-100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory-binding site at the beta subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate M(r) 5000-6000.

Animals↗
PubMed1995

Content and binding characteristics of the mitochondrial ATPase inhibitor, IF1, in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms.

We determined the IF1 contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF1-mediated inhibition of the ATPase activity of IF1-depleted rabbit heart mitochondrial particles (RHMP) by IF1-containing extracts from these same eight species. The IF1 binding experiments were run at both species-endogenous IF1 levels and at an IF1 level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF1 or either species-endogenous or normalized levels of pig heart IF1 were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF1-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF1 were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF1-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF1 inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF1 inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF1, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF1, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders.

OBJECTIVE METHODS: We describe biochemically and clinically relevant aspects of mitochondrial ATP synthase, the enzyme that supplies most ATP for the cells energy demand. RESULTS: Analyzing human Rho zero cells we could identify three subcomplexes of ATP synthase: F1 catalytic domain, F1 domain with bound natural IF1 inhibitor protein, and F1-c subcomplex, an assembly of F1 domain and a ring of F(O)-subunits c. Large amounts of F1 subcomplexes accumulated also in mitochondria of patients with specific mitochondrial disorders. By quantifying the F1 subcomplexes and other oxidative phosphorylation complexes in parallel, we were able to discriminate three classes of defects in mitochondrial biosynthesis, namely, mitochondrial DNA depletion, mitochondrial transfer RNA (tRNA) mutations, and mutations in the mitochondrial ATP6 gene. INTERPRETATION: The relatively simple electrophoretic assay used here is a straightforward approach to differentiate between various types of genetic alterations affecting the biosynthesis of oxidative phosphorylation complexes and will be useful to guide molecular genetic diagnostics in the field of mitochondrial neuromuscular disorders.

Binding Sites↗
PubMed1979

Intramitochondrial rod-like inclusions in human breast tumors.

Ultrastructural screening of human breast tumor biopsies has revealed, in 43% of the carcinomas, and in 16% of the benign lesions examined. the presence of rod-like inclusions in mitochondria of epithelial cells. The inclusions are cylindrical in cross section, with a diameter of about 50 nm, and show regular striations in longitudinal sections. They are probably derived from cristae. Mitochondria with inclusions are usually enlarged and are found most frequently in epithelial cells of invasive tumors and near zones of tumor necrosis. Similar although not identical inclusions have been observed by other investigators in the liver of women with hydatidiform mole or choriocarcinoma, of pregnant women, and of women taking oral contraceptives. Identical inclusions have been found in mitochondria of HeLa cells treated with anti-HeLa serum. Although no clear-cut correlations were found between the finding of inclusions and parameters such as age of patients, nuclear grade or lymphocytic infiltration of the breast tumors, it is conceivable that the presence of mitochondrial inclusions reflects the effect of hormonal or of immunological factors.

Adenofibroma↗
PubMed1982

Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.

The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.

ATP Synthetase Complexes↗
PubMed1984

A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus.

The major 70-kd protein of the yeast mitochondrial outer membrane is made on cytosolic ribosomes and imported into the outer membrane without proteolytic cleavage. We have attempted to identify the sequences which target the protein to the mitochondria and which permanently anchor it to the lipid bilayer of the outer membrane. By manipulating the cloned gene we have deleted 13 different regions throughout the polypeptide; in addition, we have fused amino-terminal regions of different length to beta-galactosidase. Each altered gene was introduced into yeast and the intracellular fate of the corresponding polypeptide product was determined by subcellular fractionation. All the information for targeting and anchoring the 70-kd protein (617 amino acids) was contained within the amino-terminal 41 amino acids. When this entire region was deleted, the protein was recovered with the cytosol fraction. However, several restricted deletions within this amino-terminal region appeared to affect targeting and anchoring differentially: most of the altered protein remained in the cytosol but a small fraction was misrouted into the mitochondrial matrix space. We suggest that targeting is mediated by a region which includes the 11 amino-terminal amino acids whereas the permanent membrane anchor is provided by a typical transmembrane sequence between residues 9 and 38.

Amino Acid Sequence↗
PubMed1986

Two overlapping genes in bovine mitochondrial DNA encode membrane components of ATP synthase.

Two hydrophobic proteins have been purified to homogeneity from a mixture of about 13 proteins that are extracted from bovine mitochondria with a chloroform:methanol mixture. Sequence analysis shows that the smaller is a protein of 66 amino acids and is the product of a mitochondrial gene, A6L. The larger, a protein of 226 amino acids, is ATPase-6, a membrane component of ATP synthase, also encoded in mitochondrial DNA. The protein sequences determined establish that the genes for the two proteins overlap by 40 bases and indicate that translation of the second gene, ATPase-6, is initiated within the coding region of A6L. The A6L and the ATPase-6 proteins have also been isolated from the ATP synthase complex and so appear to be bona fide components of the enzyme. The function of A6L is unknown. However, weak structural homology suggests a functional similarity to the yeast mitochondrial protein, aapI, which is required for assembly of the fungal ATP synthase complex. Homologies between ATPase-6 and subunit a of the Escherichia coli ATP synthase complex indicate that the ATPase-6 protein has a similar role in the mitochondrial complex to its bacterial counterpart, being essential for the formation of an active proton channel.

Adenosine Triphosphatases↗
PubMed1987

Transport of proteins to the mitochondrial intermembrane space: the 'matrix-targeting' and the 'sorting' domains in the cytochrome c1 presequence.

We reported earlier that the yeast cytochrome c1 presequence (length: 61 amino acids) directs attached proteins to the mitochondrial intermembrane space and that it appears to contain two functional domains: a 'matrix-targeting' domain, and a 'sorting' domain. We have now used gene manipulation together with two different in vivo import assays to map these two domains within the cytochrome c1 presequence. The 'matrix-targeting' domain is contained within the N-terminal 16 residues (or less); by itself, it directs attached proteins to the matrix. The 'sorting' domain extends into the C-terminal 13 residues of the presequence; while it does not mediate intracellular protein transport by itself, it acts together with the preceding 'matrix-targeting' sequence in sorting attached proteins into the intermembrane space. On replacing the authentic 'matrix-targeting' sequence with artificial sequences of different lengths we found that sorting of proteins between the outer membrane and the intermembrane space is not exclusively determined by the length of the N-terminal 'matrix-targeting' sequence.

Animals↗
PubMed1987

Transport of proteins to the mitochondrial intermembrane space: the 'sorting' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.

Cytochrome c Group↗
PubMed1992

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

Adenosine Triphosphate↗
PubMed1988

Studies on a possible molecular basis for the structure of mitochondrial cristae.

We have investigated a possible molecular basis for mitochondrial cristae formation. Proteoliposomes containing electron transport proteins, cytochrome oxidase, or complex III in their proper orientation bind to pig heart mitoplasts but not pig heart mitochondria. Using Leydig tumor cells, we have confirmed earlier reports that chloramphenicol causes a diminution in cristae content and a change in its characteristic lamellar form. We show that the proteoliposomes containing cytochrome oxidase or complex III in the proper orientation bind to mitoplasts from Leydig tumor cells but do not bind as well to mitoplasts from chloramphenicol-treated Leydig tumor cells. These experiments provide a possible mechanism to explain cristae formation.

Animals↗
PubMed1980

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria.

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH2-cytochrome c reductase activity with a turnover number of 15.7 sec(-1) and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c1 per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (35S)-methionine in the absence or presence of inhibitors of cytoplasmic or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using anti-serum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (35S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.

Cytochrome b Group↗
PubMed2006

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.

Animals↗