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Mulchand S Patel - One of the best experts on this subject based on the ideXlab platform.
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Binding of pyruvate dehydrogenase to the core of the human pyruvate dehydrogenase complex
FEBS Letters, 2008Co-Authors: Lioubov G Korotchkina, Mulchand S PatelAbstract:In human (h) pyruvate dehydrogenase complex (PDC) the pyruvate dehydrogenase (E1) is bound to the E1-binding domain of Dihydrolipoamide Acetyltransferase (E2). The C-terminal surface of the E1β subunit was scanned for the negatively charged residues involved in binding with E2. βD289 of hE1 interacts with K276 of hE2 in a manner similar to the corresponding interaction in Bacillus stearothermophilus PDC. In contrast to bacterial E1β, the C-terminal residue of the hE1β does not participate in the binding with positively charged residues of hE2. This latter finding shows species specificity in the interaction between hE1β and hE2 in PDC.
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r lipoic acid inhibits mammalian pyruvate dehydrogenase kinase
Free Radical Research, 2004Co-Authors: Lioubov G Korotchkina, Sukhdeep Sidhu, Mulchand S PatelAbstract:The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1 > PDK4 ∼ PDK2 > PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of Dihydrolipoamide Acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of ...
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Function of several critical amino acids in human pyruvate dehydrogenase revealed by its structure.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Lioubov G Korotchkina, Ewa Ciszak, Mulchand S PatelAbstract:Abstract Pyruvate dehydrogenase (E1), an α2β2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the Dihydrolipoamide Acetyltransferase. The roles of βW135, αP188, αM181, αH15, and αR349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: βW135 is in the center of the hydrophobic interaction between β and β′ subunits; αP188 and αM181 are critical for the conformation of the TPP-binding motif and interaction between α and β subunits; αH15 is necessary for the organization of the N-terminus of α and α′ subunits; and αR349 supports the interaction of the C-terminus of the α subunits with the β subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.
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Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase
Journal of Biological Chemistry, 2003Co-Authors: Ewa Ciszak, Lioubov G Korotchkina, Sukhdeep Sidhu, Paulina M. Dominiak, Mulchand S PatelAbstract:Abstract The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In α2β2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Cα-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-Dihydrolipoamide Acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted ∼2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.
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Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase
2003Co-Authors: Ewa Ciszak, Lioubov G Korotchkina, Paulina M. Dominiak, Sukdeep Sidhu, Mulchand S PatelAbstract:The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-Dihydrolipoamide Acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.
Mallavarapu Megharaj - One of the best experts on this subject based on the ideXlab platform.
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pyrene degradation by chlorella sp mm3 in liquid medium and soil slurry possible role of Dihydrolipoamide Acetyltransferase in pyrene biodegradation
Algal Research-Biomass Biofuels and Bioproducts, 2017Co-Authors: Suresh R Subashchandrabose, Panneerselvan Logeshwaran, Kadiyala Venkateswarlu, Ravi Naidu, Mallavarapu MegharajAbstract:Abstract Microalgae inhabiting the real contaminated sites are capable of degrading organic pollutants. In the present study, the potential of a microalga, Chlorella ssp. MM3, a soil isolate from a former cattle dip site, was assessed in degrading pyrene both in aqueous medium and soil slurry. Strain MM3 can grow on pyrene in culture medium at concentrations as high as 250 μM. When grown in presence of 50 μM pyrene, the cell density increased from 1.1 × 105 cells mL− 1 to 16.45 × 105 cells mL− 1 within 7 days. With an initial cell density of 3 × 107 cells mL− 1, nearly 70% of 50 μM pyrene was degraded after 7 days of incubation. When compared with Triton X-100, Tween 80 was a better non-ionic surfactant for pyrene biodegradation. Nearly 20% increase in degradation of pyrene was observed with the use of 0.005% Tween 80. Differential protein expression in pyrene-grown cells of the microalga resulted in distinct accumulation of Dihydrolipoamide Acetyltransferase (or dihydrolipoyl transacetylase), one of the three components of pyruvate dehydrogenase complex, indicating a possible role of this enzyme in microalgal degradation of pyrene. The microalgal cells immobilized in calcium alginate completely degraded 50 μM of pyrene within 10 days in nonsterile soil slurry treated with 0.005% Tween 80. Our results clearly indicate that the strain MM3 has a great potential for its use in remediating soils contaminated with pyrene.
Lioubov G Korotchkina - One of the best experts on this subject based on the ideXlab platform.
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Binding of pyruvate dehydrogenase to the core of the human pyruvate dehydrogenase complex
FEBS Letters, 2008Co-Authors: Lioubov G Korotchkina, Mulchand S PatelAbstract:In human (h) pyruvate dehydrogenase complex (PDC) the pyruvate dehydrogenase (E1) is bound to the E1-binding domain of Dihydrolipoamide Acetyltransferase (E2). The C-terminal surface of the E1β subunit was scanned for the negatively charged residues involved in binding with E2. βD289 of hE1 interacts with K276 of hE2 in a manner similar to the corresponding interaction in Bacillus stearothermophilus PDC. In contrast to bacterial E1β, the C-terminal residue of the hE1β does not participate in the binding with positively charged residues of hE2. This latter finding shows species specificity in the interaction between hE1β and hE2 in PDC.
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r lipoic acid inhibits mammalian pyruvate dehydrogenase kinase
Free Radical Research, 2004Co-Authors: Lioubov G Korotchkina, Sukhdeep Sidhu, Mulchand S PatelAbstract:The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1 > PDK4 ∼ PDK2 > PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of Dihydrolipoamide Acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of ...
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Function of several critical amino acids in human pyruvate dehydrogenase revealed by its structure.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Lioubov G Korotchkina, Ewa Ciszak, Mulchand S PatelAbstract:Abstract Pyruvate dehydrogenase (E1), an α2β2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the Dihydrolipoamide Acetyltransferase. The roles of βW135, αP188, αM181, αH15, and αR349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: βW135 is in the center of the hydrophobic interaction between β and β′ subunits; αP188 and αM181 are critical for the conformation of the TPP-binding motif and interaction between α and β subunits; αH15 is necessary for the organization of the N-terminus of α and α′ subunits; and αR349 supports the interaction of the C-terminus of the α subunits with the β subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.
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Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase
Journal of Biological Chemistry, 2003Co-Authors: Ewa Ciszak, Lioubov G Korotchkina, Sukhdeep Sidhu, Paulina M. Dominiak, Mulchand S PatelAbstract:Abstract The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In α2β2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Cα-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-Dihydrolipoamide Acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted ∼2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.
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Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase
2003Co-Authors: Ewa Ciszak, Lioubov G Korotchkina, Paulina M. Dominiak, Sukdeep Sidhu, Mulchand S PatelAbstract:The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-Dihydrolipoamide Acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.
Richard N. Perham - One of the best experts on this subject based on the ideXlab platform.
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protein protein interactions in the pyruvate dehydrogenase multienzyme complex Dihydrolipoamide dehydrogenase complexed with the binding domain of Dihydrolipoamide Acetyltransferase
Structure, 1996Co-Authors: Sharmila S Mande, Richard N. Perham, Steve Sarfaty, Mark D Allen, Wim G J HolAbstract:Abstract Background: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of Dihydrolipoamide Acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and Dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). Results In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 a resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. Conclusion Combining this new structure for the E3–E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex.
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Protein–protein interactions in the pyruvate dehydrogenase multienzyme complex: Dihydrolipoamide dehydrogenase complexed with the binding domain of Dihydrolipoamide Acetyltransferase
Structure, 1996Co-Authors: Sharmila S Mande, Richard N. Perham, Steve Sarfaty, Mark D Allen, Wim G J HolAbstract:Abstract Background: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of Dihydrolipoamide Acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and Dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). Results In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 a resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. Conclusion Combining this new structure for the E3–E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex.
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the high resolution structure of the peripheral subunit binding domain of Dihydrolipoamide Acetyltransferase from the pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus
Journal of Molecular Biology, 1994Co-Authors: Yogeshvar N Kalia, Ettore Appella, D S Hipps, Simon M Brocklehurst, Kazuyasu Sakaguchi, Richard N. PerhamAbstract:Abstract The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of Dihydrolipoamide Acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1 H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (φ,χ 1 ) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0·36 A for the backbone atoms and 0·96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel α-helices (residues Val7 to Lys14 and Lys32 to Leu39), a 3 10 -helix (residues Asp17 to Val21) and a structured loop made up of overlapping β-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residue, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.
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Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the Dihydrolipoamide Acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.
Biochemical Journal, 1992Co-Authors: D S Hipps, Richard N. PerhamAbstract:A sub-gene encoding the N-terminal 170 residues of the Dihydrolipoamide Acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the Dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.
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Two lipoyl domains in the Dihydrolipoamide Acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis.
FEBS Letters, 1991Co-Authors: Andrew G. Allen, Richard N. PerhamAbstract:Abstract A fragment of DNA incorporating the gene, pdhC . that encodes the Dihydrolipoamide Acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis was cloned and a DNA sequence of 2360 bp was determined. The pdhC gene (1620 bp) corresponds to an E2 chain of 539 amino acid residues. M r 56 466, comprising two lipoyl domains, a peripheral subunit-binding domain and an Acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, proline and charged amino acids. The S. faecalis E2 chain differs in the number of its lipoyl domains from the E2 chains of all bacterial pyruvate dehydrogenase complexes hitherto described.
James Neuberger - One of the best experts on this subject based on the ideXlab platform.
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Immunopathology of primary biliary cirrhosis.
European Journal of Gastroenterology & Hepatology, 1999Co-Authors: R Joplin, James NeubergerAbstract:A major advance in the study of primary biliary cirrhosis was identification of the major B-cell auto-antigen as the mitochondrial enzyme pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase (PDC-E2). Subsequent studies revealed that PDC-E2 also contained epitopes recognized by patients' T cells. Furthermore, aberrant expression of MHC class II, intercellular adhesion molecules, lymphocyte co-stimulatory molecules and B-cell epitopes of PDC-E2 was observed on patients' biliary epithelium, supporting the concept that biliary epithelial cells are the target of a focused autoimmune reaction. Changes in distribution of auto-antigen on biliary epithelium and the presence of auto-antibody in patient's serum have both been shown to occur very early in the natural history of primary biliary cirrhosis, suggesting an intimate role for these molecules in immunopathogenetic mechanisms.
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Subcellular localization of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase in human intrahepatic biliary epithelial cells
The Journal of Pathology, 1995Co-Authors: R Joplin, Gerald D. Johnson, J. Gordon Lindsay, Alastair J. Strain, Stephen J. Yeaman, Lorraine Wallace, Jeremy M. Palmer, James NeubergerAbstract:In previous histological studies, biliary epithelial cells (BEC) in the liver of patients with primary biliary cirrhosis (PBC), but not controls, reacted strongly with antibodies specific for the major autoantigen associated with PBC, the E2 component of pyruvate dehydrogenase complex (PDC-E2). In this study we have used transmission electron microscopy (TEM) to document the precise subscellular localization of PDC-E2 in BEC. Two antibodies which recognize PDC-E2 were used : affinity-purified anti-PDC-E2 raised in rabbits ; and human antibody from the serum of patients with PBC, affinity-purified against human heart PDC. The intracellular localization of antibody binding was determined by laser scanning confocal microscopy and TEM. Both antibodies bound to the inner membrane of mitochondria in BEC isolated from both patients with PBC and controls, but binding to the external aspect of the plasma membrane was observed only in BEC from patients with PBC. Surface antigen expression in PBC may make BEC immunological targets.
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distribution of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase pdc e2 and another mitochondrial marker in salivary gland and biliary epithelium from patients with primary biliary cirrhosis
Hepatology, 1994Co-Authors: R Joplin, Gerald D. Johnson, John B. Matthews, John Hamburger, Stefan G. Hubscher, Alastair J. Strain, Gordon J Lindsay, James NeubergerAbstract:Previous studies in which quantitative immunofluorescence was used have shown that certain biliary epithelial cells in liver with primary biliary cirrhosis show increased levels of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase compared with controls. This study was designed to determine whether the increase in intensity of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase in biliary epithelial cells is accounted for by an increase in the number of mitochondria in the same cells. A double-antibody staining technique was used with antibodies specific for pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase and another mitochondrial inner membrane marker, recognized by the mouse monoclonal antibody MCA151A. Distribution of the antigens was studied in sections of liver and salivary gland, an additional site that is frequently involved in primary biliary cirrhosis. Confocal microscopy was used to quantify the intensity of fluorescence resulting from binding of fluorochrome-labeled antibody. In both liver and salivary glands MCA151A binding was similar in normal and sections with primary biliary cirrhosis and corresponded to the predicted distribution of mitochondria in these tissues. In the liver staining was less intense in biliary epithelial cells than in hepatocytes. In salivary gland binding of both antibodies was predominantly localized to duct cells, with those forming striated ducts, known to be rich in mitochondria, being most intensely stained. There was high coincidence of the two antigens in salivary glands (p < 0.01) and in biliary epithelial cells from normal liver (p = 0.01). However, in liver with primary biliary cirrhosis, despite high coincidence between the antigens on hepatocytes, biliary epithelial cells showed high intensity of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase but not MCA151A.(ABSTRACT TRUNCATED AT 250 WORDS)
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Distribution of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase (PDC‐E2) and another mitochondrial marker in salivary gland and biliary epithelium from patients with primary biliary cirrhosis
Hepatology, 1994Co-Authors: R Joplin, Gerald D. Johnson, John B. Matthews, John Hamburger, J. Gordon Lindsay, Stefan G. Hubscher, Alastair J. Strain, James NeubergerAbstract:Previous studies in which quantitative immunofluorescence was used have shown that certain biliary epithelial cells in liver with primary biliary cirrhosis show increased levels of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase compared with controls. This study was designed to determine whether the increase in intensity of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase in biliary epithelial cells is accounted for by an increase in the number of mitochondria in the same cells. A double-antibody staining technique was used with antibodies specific for pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase and another mitochondrial inner membrane marker, recognized by the mouse monoclonal antibody MCA151A. Distribution of the antigens was studied in sections of liver and salivary gland, an additional site that is frequently involved in primary biliary cirrhosis. Confocal microscopy was used to quantify the intensity of fluorescence resulting from binding of fluorochrome-labeled antibody. In both liver and salivary glands MCA151A binding was similar in normal and sections with primary biliary cirrhosis and corresponded to the predicted distribution of mitochondria in these tissues. In the liver staining was less intense in biliary epithelial cells than in hepatocytes. In salivary gland binding of both antibodies was predominantly localized to duct cells, with those forming striated ducts, known to be rich in mitochondria, being most intensely stained. There was high coincidence of the two antigens in salivary glands (p < 0.01) and in biliary epithelial cells from normal liver (p = 0.01). However, in liver with primary biliary cirrhosis, despite high coincidence between the antigens on hepatocytes, biliary epithelial cells showed high intensity of pyruvate dehydrogenase Dihydrolipoamide Acetyltransferase but not MCA151A.(ABSTRACT TRUNCATED AT 250 WORDS)
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membrane Dihydrolipoamide Acetyltransferase e2 on human biliary epithelial cells in primary biliary cirrhosis
The Lancet, 1992Co-Authors: R Joplin, J. G. Lindsay, Alastair J. Strain, James Neuberger, Gerald D. JohnsonAbstract:Primary biliary cirrhosis (PBC) is associated with serum antibodies that react with the Dihydrolipoamide Acetyltransferase component (E2) of the mitochondrial pyruvate dehydrogenase complex. We have sought the presence of E2 on the surface of human intrahepatic biliary epithelial cells (BEC). Cultured BECs from PBC but not normal liver were found to have E2 on the membrane after three days' culture. Isolated, viable cells examined by laser-scanning confocal microscopy revealed the pattern of E2 staining on the membrane to be similar to that seen with the membrane glycoprotein marker, HEA-125. By contrast, BECs from normal liver showed membrane staining only with HEA-125. When BECs were fixed before incubation with antibody to E2, cytoplasmic staining was observed. Our results suggest that E2 is present on the surface of biliary epithelial cells in PBC, and support the idea of a pathogenetic association between antimitochondrial antibodies and bileduct damage.