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R A Capaldi

Publications and source records attributed to R A Capaldi.

At least 145 records · Page 8Linked to original sources
PubMed1984

Lactic acidosis and mitochondrial myopathy associated with deficiency of several components of complex III of the respiratory chain.

We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions. The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patient's muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 mumol/min/g tissue in whole muscle (controls 1.16 +/- 0.47 mumol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patient's mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone. Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1983

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments.

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.

Animals↗
PubMed1983

Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase.

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.

Animals↗
PubMed1983

Deficiency in ubiquinone cytochrome c reductase in a patient with mitochondrial myopathy and lactic acidosis.

The skeletal muscle of a patient with a mitochondrial myopathy was examined. A defect in the electron transport chain was identified at the position of complex III by activity measurements and the low levels of reducible cytochrome b. The polypeptide composition of complex III in the patient's mitochondria was determined by antibody binding experiments. The method allowed detection of individual polypeptides at a lower limit of 10-40 ng of protein. Characterization of protein composition is thus possible by using a biopsy sample of 1 g of tissue. The level of core proteins, FeS protein, and subunit VI was greatly diminished in the patient's mitochondria. Cytochrome c1 polypeptide was found at normal levels but was sensitive to proteolysis by trypsin. These results show that complex III is not assembled in the patient's mitochondria. The possible role of cytochrome b as the site of the primary lesion is discussed.

Acidosis↗
PubMed1982

Cytochrome c is cross-linked to subunit II of cytochrome c oxidase by a water-soluble carbodiimide.

Modification of beef heart cytochrome c oxidase with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) has been found to significantly inhibit the high-affinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Vmax resulted in a 16-fold increase in the Km for cytochrome c. The loss in activity was accompanied by modification of subunit II to form a new species, II', which migrated somewhat more rapidly than the unmodified subunit during sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. This new species was the major site of radiolabeling when cytochrome c oxidase was treated with [14C]CH3EDC, indicating covalent incorporation of the carbodiimide. Equimolar concentrations of cytochrome c dramatically protected cytochrome c oxidase from inhibition by the carbodiimide and in approximately the same proportion shielded subunit II from modification to the labeled II' species. In addition, cytochrome c was cross-linked to subunit II to form a new species migrating somewhat faster than subunit I during NaDodSO4 gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged N-acylurea which inhibits cytochrome c binding. In the presence of cytochrome c, EDC promotes formation of amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.

Animals↗
PubMed1982

Preparation of two-dimensional arrays from purified beef heart cytochrome c oxidase.

A method for preparing two-dimensional crystals from highly purified beef heart cytochrome c oxidase is described. This involves mixing the enzyme with phosphatidylcholine and then extracting excess lipid with deoxycholate. The reconstituted crystals show PI symmetry. Alternating rows of monomers are more closely packed (along the b axis) than in the previously described P121 crystals. However, the monomer structure in projection is the same in the two crystal forms. The P121 crystal form has been reacted with trypsin. This treatment did not alter the crystals but removed most of the impurities present in these cytochrome c oxidase preparation of low purity.

Animals↗
PubMed1981

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide.

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.

Amino Acid Sequence↗
PubMed1981

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase.

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.

Amino Acids↗
PubMed1981

Interaction of succinate--ubiquinone reductase (complex II) with (arylazido)phospholipids.

The interaction of purified succinate dehydrogenase and succinate--ubiquinone reductase (complex II) with lipids was explored by using two (arylazido)phospholipids, one with the reactive nitrene in the head-group region of the bilayer [1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (PLII)] and one with the nitrene on the methyl terminus of one of the fatty acid chains [1-myristoyl-2-[12-[(2-azido-4-nitrophenyl)amino]lauroyl]-sn-glycero-3-[14C]phosphocholine (PLI)]. Protein was reacted with vesicles of egg lecithin containing radioactive (arylazido)-phospholipids and the covalent cross-linking of lipid and protein induced by irradiation under UV light. Purified succinate dehydrogenase was found to bind to lipid vesicles through both subunits as both were labeled by PLII. The smaller subunit was inserted into the interior of the bilayer and labeled by PLI. Complex II was found to interact with lipid vesicles, with the smaller subunit of succinate dehydrogenase, CII-3, and CII-4 all inserted into the interior of the bilayer. The large subunit of succinate dehydrogenase was found to be held above the bilayer in complex II and not labeled by either probe. Results are used to derive a picture of the arrangement of subunits in complex II and to evaluate the utility of (arylazido)-phospholipids in membrane studies.

Affinity Labels↗