An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the... more
An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be ...
Research Interests: Biochemistry, Kinetics, Biology, Medicine, Biological Sciences, and 12 moreCell line, Animals, Peptides, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Immunoprecipitation, CHEMICAL SCIENCES, Cattle, Chemical Precipitation, Biosynthesis, Polyclonal Antibodies, and Medical and Health Sciences
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1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2–5 times more active with added native DNA as template and the supernatant... more
1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2–5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50μg of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg2+ and K+ concentrations. 6. Maximal enzyme activity is approached with 40μg of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with ...
Research Interests: Biochemistry, Molecular Biology, Biology, DNA replication, Medicine, and 15 moreCell Division, Biological Sciences, DNA, Cell line, Mice, Animals, Biochemical, Enzyme, nuclear DNA, CHEMICAL SCIENCES, Cell nucleus, Analytical Ultracentrifugation, Cytoplasm, DNA Polymerase, and Medical and Health Sciences
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Research Interests: Chemistry, Medicine, Mutagenesis, Icon, Citation, and 11 moreDownload, Protein Secondary Structure Prediction, Substrate Specificity, Bacillus megaterium, Cysteine, ICON, Amino Acid Sequence, Site-directed Mutagenesis, Biochemistry and cell biology, conserved sequence , and Medical biochemistry and metabolomics
1. In primary biliary cirrhosis, the major M2 autoantigen, reacting with antimitochondrial antibodies in sera from >90% of patients, has been identified as the E2 component of the pyruvate dehydrogenase complex. However, two recent... more
1. In primary biliary cirrhosis, the major M2 autoantigen, reacting with antimitochondrial antibodies in sera from >90% of patients, has been identified as the E2 component of the pyruvate dehydrogenase complex. However, two recent reports suggest that alternative polypeptides may be major autoantigens. 2. The evidence that a 75 kDa subunit of complex I of the respiratory chain is a major autoantigen (Frostell, Mendel-Hartvig, Nelson, Totterman, Bjorkland & Ragan, Scand. J. Immunol. 1988; 28, 157–65) is refuted. The findings of Frostell et al. can be explained by contamination of complex I with the pyruvate dehydrogenase complex, evidence for which is presented here. 3. Inspection of the partial amino acid sequence of an unidentified mitochondrial autoantigen (Muno, Kominami, Ishii, Usui, Saituku, Sakakibara & Namihisa, Hepatology 1990; 11, 16–23) shows that it is the El β-subunit of the pyruvate dehydrogenase complex, previously identified as a major autoantigen, and not a ‘new’...
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The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their... more
The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge ...
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Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not... more
Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 wit...
Research Interests: Biochemistry, Biology, Medicine, Biological Sciences, Magnesium, and 12 moreAnimals, Phosphorylation, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Cattle, Dehydrogenase, Myocardium, Substrate Specificity, Hydrolysis, proteolysis, and Medical and Health Sciences
The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane... more
The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest ...
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Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1... more
Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly. Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e. 8–12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e. 50-fold) of parent E3. N-terminal sequence analysis of the truncated 35000-Mr protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15000-Mr N-terminal fragment comprising both the lipoyl and linker sequences. In...
Research Interests: Biochemistry, Kinetics, Biology, Medicine, Biological Sciences, and 13 moreAnimals, Peptides, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Cattle, Dehydrogenase, Myocardium, Molecular weight, Amino Acid Sequence, Hydrogen-Ion Concentration, Molecular Sequence Data, and Medical and Health Sciences
The ‘Covalent Switching’ hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc.... more
The ‘Covalent Switching’ hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem pr...
Research Interests: Chemistry, Catalysis, Medicine, Biological Sciences, Mutation, and 15 moreCircular Dichroism, Escherichia coli, Electron Transfer, Stereochemistry, Biochemical, Electron Transport, Tryptophan, Heme, CHEMICAL SCIENCES, Phenylalanine, Bacillus megaterium, Alanine, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
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The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M)... more
The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms h...
Research Interests: Chemistry, Protein Folding, Fluorescence, Medicine, Biological Sciences, and 13 moreCircular Dichroism, Animals, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Gel Permeation Chromatography, Enzyme, CHEMICAL SCIENCES, Cattle, Protein Secondary Structure Prediction, Myocardium, Guanidines, Medical and Health Sciences, and Guanidinium Chloride
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Summary Large single crystals (up to 1 mm in each dimension) of the B800–850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of β-octyl-glucoside. These crystals have the space group R32 and... more
Summary Large single crystals (up to 1 mm in each dimension) of the B800–850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of β-octyl-glucoside. These crystals have the space group R32 and unit cell dimensions of a = b = 119.9 Å and c = 297.0 Å. Recently we have improved our crystallization procedures so that all crystals now diffract reliably to beyond 3.5 Å, with some diffracting to below 3 Å. A range of isomorphous heavy atom derivatives have been prepared and we are now engaged in locating the heavy atom sites within the unit cell.
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... It is now clear that we have a reasonably detailed picture of the structure and the function of the purple-bacterial PSU. Can this provide clues to help design better solar cells? The indications are that this new knowledge is... more
... It is now clear that we have a reasonably detailed picture of the structure and the function of the purple-bacterial PSU. Can this provide clues to help design better solar cells? The indications are that this new knowledge is providing ...
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Publisher Summary The B800-850 antenna complex from Rps, sphaeroides is one of the best characterized of the bacterial antenna complexes. This complex is a hydrophobic, integral membrane protein and probably exists in vivo as aggregates... more
Publisher Summary The B800-850 antenna complex from Rps, sphaeroides is one of the best characterized of the bacterial antenna complexes. This complex is a hydrophobic, integral membrane protein and probably exists in vivo as aggregates of a well-defined minimal unit. This minimal unit consists of three molecules of Bchla and one molecule of Car non-covalently bound to two low molecular weight polypeptides. Two of these Bchla molecules give rise to the 850 nm absorption band, while the third is responsible for the 800 nm absorption. This chapter discusses the membrane location of this antenna complex using antibodies prepared to the isolated, purified B800–850. There are antigenic sites recognized by the antiserum to the B800–850 antenna complex present on both sides of the photosynthetic membrane of Rps, sphaeroides.
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Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. The resultant types... more
Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. The resultant types of chemical damage to lipids, DNA and proteins are described as well as the broader implications for the involvement of ROS in disease onset and progression. The relative contributions of mitochondrial, enzyme-linked, antioxidant defence systems to tissue protection are also reviewed as is the emerging importance of the peroxiredoxin family in general to H2O2-mediated signalling The constituent enzymes of the mitochondrial PrxIII pathway are discussed in detail including the roles of PrxIII and PrxV in their capacities as typical 2-cys and atypical 2-cys thioredoxin-dependent hydroperoxide reductases, respectively. The structures and catalytic mechanisms of PrxIII and V are examined and some key properties of the reconstituted mitochondrial PrxIII pathway are highlighted with specific reference to the susceptibility of peroxiredoxins to inactivation at elevated H2O2 levels and their potential for participation in H2O2-mediated signalling responses. It is concluded that mitochondrial Prxs form a vital link in an integrated cellular antioxidant defence network that minimises ROS-mediated damage and ensures that cells mount appropriate responses to increased levels of oxidative stress via the upregulation of key cell signalling pathways.
Research Interests: Cell Biology, Oxidative Stress, Mitochondria, Medicine, Catalytic Mechanism, and 15 moreHumans, Reactive Oxygen Species, Animals, Antioxidant, Cell Signalling, Mitochondrial Respiratory Chain, Electron Transport, Enzyme, Hydrogen Peroxide, Peroxiredoxin, Oxidative phosphorylation, Oxygen Consumption, Peroxiredoxins, Mitochondrion, and Mitochondrial Proteins
Research Interests: Biochemistry, Biology, Phospholipids, Calcium, Medicine, and 15 moreCarbon Isotopes, Animals, Cattle, Rats, Time Factors, Bovine Serum Albumin, Thin Layer Chromatography, Phosphatidylcholine, Oxygen Consumption, Edetic Acid, Biochemistry and cell biology, Mitochondrion, Rat Liver, Medical biochemistry and metabolomics, and In Vitro Techniques
The recently characterized Mr-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component... more
The recently characterized Mr-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.
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The mammalian pyruvate dehydrogenase complex, Mr 8.5 X 10(6), contains an additional tightly bound 50 000-Mr polypeptide, component X, which copurifies with the intact assembly. Small amounts of the individual E2 and X polypeptides were... more
The mammalian pyruvate dehydrogenase complex, Mr 8.5 X 10(6), contains an additional tightly bound 50 000-Mr polypeptide, component X, which copurifies with the intact assembly. Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with 125I-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-14C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These protein-chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70 000) subunit. Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.
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Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme... more
Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross‐linked by reaction with phenylene‐o‐bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross‐linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of d...
Research Interests: Biochemistry, Biology, Medicine, Female, Animals, and 15 moreMale, Peptides, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Gel Permeation Chromatography, Cattle, European, Rats, Myocardium, Densitometry, Structure activity Relationship, Protein Binding, Structure Function, Biochemistry and cell biology, binding sites, and Medical biochemistry and metabolomics
Research Interests: Chemistry, Crystallography, Structure, Medicine, Macromolecular X-Ray Crystallography, and 14 moreBiological Sciences, Mutation, Animals, CHEMICAL SCIENCES, Cattle, Catenane, Catenanes, Peroxiredoxin, Cysteine, Dimer, Protein Quaternary Structure, Dimerization, Peroxiredoxins, and Mitochondrial Proteins
The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein... more
The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH 2 -terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the α-ketoglutarate dehydrogenase complex (αKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of αKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo . Covalent modification of αKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 i...
Research Interests: Biochemistry, Biology, Mitochondria, Medicine, Multidisciplinary, and 7 moreHSP, Kidney, Animals, Cell Death, Rats, Heat Shock Proteins, and Cysteine
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The antenna complexes from Rps. cryptolactis have been isolated and purified. Rps. cryptolactis contains two types of variable antenna complex, B800-850 and B800-820 as well as the 'core' B875 antenna complex. The variable... more
The antenna complexes from Rps. cryptolactis have been isolated and purified. Rps. cryptolactis contains two types of variable antenna complex, B800-850 and B800-820 as well as the 'core' B875 antenna complex. The variable antenna complexes contain more than two types of antenna apoprotein, and have a Bchla:carotenoid ratio of ∼2:1. They can both be crystallised, but the B800-820 complex is the easiest with which to get relatively large single 3-D crystals (up to 0.5 mm in each dimension).
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This review sets out the case that now is the time for plant science to establish the technologies required for routinely studying the structure and function of plant proteins. The impact that protein structural information can have is... more
This review sets out the case that now is the time for plant science to establish the technologies required for routinely studying the structure and function of plant proteins. The impact that protein structural information can have is illustrated here with reference to photosynthesis. Our understanding of the precise molecular mechanisms of the light‐reactions of photosynthesis has been transformed by the combination of high‐resolution protein structural data and detailed functional studies. The past few years have been a particularly exciting time to be engaged in basic plant science research. The application of modern techniques of molecular biology has allowed many key questions to be addressed. The stage is now set for an even bigger revolution as the current plant genome sequencing projects are completed. If these advances are going to be fully exploited, plant science must get to grips with studying proteins, not just genes. Reliable methods for the overexpression of proteins...
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Research Interests: Endocrinology, Neuroscience, Biology, Progressive Supranuclear Palsy, Oxidative Stress, and 15 moreMitochondria, Energy Metabolism, Humans, Internal Medicine, Cerebellum, Mice, Nitric oxide, Animals, Mitochondrial Diseases, Neurobiology of Aging, Neurosciences, Glutamate Dehydrogenase, Malondialdehyde, Immunoblotting, and Aconitase
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Research Interests: Chemistry, Fluorescence Spectroscopy, Molecular Biology, EM, Medicine, and 15 moreCrystal structure, Humans, Circular Dichroism, Enzyme, Analytical Ultracentrifugation, Cryo Electron Microscopy, CTD, Amino Acid Sequence, Low Resolution, PDB, Full Length Movies, Biochemistry and cell biology, Molecular Sequence Data, binding sites, and cryo EM
Research Interests: Molecular Biology, Biology, Medicine, Molecular, Cell Differentiation, and 15 moreHumans, Mutation, Phage display, Monoclonal Antibodies, Molecular Mimicry, B Lymphocytes, Monoclonal Antibody, Amino Acid Sequence, Epitopes, Enzyme Linked Immunosorbent Assay, Autoantibodies, Biochemistry and cell biology, Molecular Sequence Data, epitope, and B cell
Research Interests: Chemistry, Electron Microscopy, Catalysis, Mitochondria, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Mutation, Circular Dichroism, Escherichia coli, Animals, Biological, CHEMICAL SCIENCES, Cattle, Molecular cloning, Peroxiredoxin, Peroxidases, Cysteine, Histidine, and Medical and Health Sciences
Research Interests: Protein Folding, Catalysis, Biology, Biological Chemistry, Medicine, and 12 moreBiological Sciences, Escherichia coli, Animals, Biological, Trypsin, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Cattle, Elsevier, Structure activity Relationship, Dimerization, and Medical and Health Sciences
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, X Rays, and 12 moreHumans, Animals, Biological, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Analytical Ultracentrifugation, Species Specificity, Protein Binding, Dimerization, binding sites, and Medical and Health Sciences
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The reason for the close association between primary biliary cirrhosis and the appearance of antibodies that recognize the E2 component of pyruvate dehydrogenase complex is not understood. The distribution of the three pyruvate... more
The reason for the close association between primary biliary cirrhosis and the appearance of antibodies that recognize the E2 component of pyruvate dehydrogenase complex is not understood. The distribution of the three pyruvate dehydrogenase complex subunits was examined in the liver and lymph nodes of patients with primary biliary cirrhosis, patients with other liver diseases and normal subjects by immunohistochemistry using affinity-purified antibodies. Intensity of staining was assessed semiquantitatively and validated by scanning laser confocal microscopy. In primary biliary cirrhosis tissue, the E2 staining pattern did not parallel the reported distribution of mitochondria. E2 staining in biliary epithelial cells was consistently stronger than in hepatocytes. In primary biliary cirrhotic liver, staining of biliary epithelium was significantly stronger than in normal or other liver disease controls; many bile ducts in primary biliary cirrhotic liver demonstrated very high intensity, diffuse distribution of stain. No differences in staining intensity were seen between perivenular hepatocytes in primary biliary cirrhotic liver and those in controls; periportal hepatocytes in primary biliary cirrhotic liver were, however, more intensely stained than perivenular cells. In primary biliary cirrhotic portal lymph nodes, a subset of macrophages showed high-intensity, diffuse distribution of stain. By contrast, staining with antibodies to E1 and E3 (other components of pyruvate dehydrogenase complex) produced uniform-intensity, mitochondrial distribution both in primary biliary cirrhosis and control tissue. The increased intensity of E2 in primary biliary cirrhotic tissue could be explained in terms of abnormal metabolism of E2 by biliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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In brain cells, various metabolites and metabolic pathways, largely of mitochondrial origin, have been shown to be compartmentalized. Attention has therefore been focused on the possible existence of mitochondrial heterogeneity in the... more
In brain cells, various metabolites and metabolic pathways, largely of mitochondrial origin, have been shown to be compartmentalized. Attention has therefore been focused on the possible existence of mitochondrial heterogeneity in the brain at the cellular level. To determine whether mitochondria in cultured cortical and cerebellar astrocytes are heterogeneous at the single cell level, immunogold electron microscopy and an antibody against the alpha-ketoglutarate dehydrogenase component of the alpha-ketoglutarate dehydrogenase complex, a marker enzyme for the tricarboxylic acid (TCA) cycle, were employed. The number of gold particles was counted in the mitochondria of 36 and 42 cells from cultured cerebellar and cortical astrocytes, respectively. A test for random distribution (Poisson distribution) of mitochondria according to the number of gold particles was subsequently performed for every one of the 36 and 42 cells as the ratio variance/mean (= index of dispersion). This should be approximately distributed as chi2/degrees of freedom (df) = n - 1, n = number of mitochondria), if the observations obeyed a Poisson distribution. For 26 of the 36 (cerebellar astrocytes) distributions and for 28 of the 42 (cortical astrocytes) distributions a random distribution had to be rejected. These findings therefore strongly indicate that alpha-ketoglutarate dehydrogenase is heterogeneously distributed in mitochondria within individual astrocytes originating either from cerebellum or cerebral cortex. In conclusion, this study underlines the probability that mitochondrial heterogeneity at the single cell level might be extended to involve other metabolic pathways and metabolites.
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An intact B890 light‐harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low‐M r polypeptide. Both these polypeptides are present in the intact... more
An intact B890 light‐harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low‐M r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.
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The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose‐6‐phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein.... more
The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose‐6‐phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein. The T2 protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having type 1c glycogen storage disease.
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The interactions of the individually expressed haem‐ and flavin‐containing domains of cytochrome P‐450 BM3 have been analysed by enzymological and spectroscopic techniques. Electron transfer between the isolated domains occurs at a much... more
The interactions of the individually expressed haem‐ and flavin‐containing domains of cytochrome P‐450 BM3 have been analysed by enzymological and spectroscopic techniques. Electron transfer between the isolated domains occurs at a much lower rate than that occurring in the intact flavocytochrome. CD spectroscopic studies indicate that the linkage of the domains in intact P‐450 BM3 creates haem and amino acid environments suitable for efficient electron transfer from its flavin domain.