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1.
J Parasitol ; 105(2): 321-329, 2019 04.
Article in English | MEDLINE | ID: mdl-30998130

ABSTRACT

The mitochondrial, inner-membrane-associated, reversible NADPH→NAD+ transhydrogenase of the energetically anaerobic adult cestode Hymenolepis diminuta connects NADPH generation, via a mitochondrial NADP+-specific "malic" enzyme, with NADH formation needed for electron transport. In reducing the pyridine nucleotide, the enzyme concomitantly catalyzes transmembrane proton translocation, thereby coupling NADH formation to ATP generation or NADPH formation to ATP hydrolysis. Detergent-solubilized transhydrogenase, from isolated mitochondrial membranes, was purified to apparent homogeneity using ion exchange and hydroxylapatite chromatographies. The enzyme displayed a monomeric Mr of ∼110 kDa and required phospholipid, without which activity was rapidly lost. Of the phospholipids examined, phosphatidylcholine was the most effective. Transhydrogenase-catalyzed NADH formation was inhibited by NAD(P)+ and adenylates, suggesting regulatory effects of the pyridine nucleotides and effects of pyridine nucleotide-simulating molecules. In keeping with its proton-translocating function, the enzyme was inhibited by dicyclohexylcarbodiimide. The isolated enzyme catalyzed neither NADH→NADP+ nor NADH→NAD+ transhydrogenations, thereby suggesting a need for a minimal coupling to electron transport for the NADH→NADP+ reaction as well as enzyme specificity. Anti-transhydrogenase monospecific antibodies proved inhibitory to NADPH→NAD+ transhydrogenation catalyzed by both isolated and membrane-associated enzymes. This purification study apparently represents a first for parasitic helminths or multicellular invertebrates generally and establishes a framework for evaluating the transhydrogenase as a potential site for specific chemotherapeutic attack.


Subject(s)
Hymenolepis diminuta/enzymology , Mitochondria/enzymology , NADP Transhydrogenases/isolation & purification , NADP/metabolism , NAD/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Female , Immunoblotting , Immunoglobulin G/immunology , Male , NADP Transhydrogenases/antagonists & inhibitors , NADP Transhydrogenases/immunology , NADP Transhydrogenases/metabolism , Phospholipids/metabolism , Phospholipids/pharmacology , Rabbits , Rats , Rats, Sprague-Dawley
2.
Eur J Biochem ; 267(11): 3281-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10824114

ABSTRACT

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the alpha and beta subunits remained tightly associated. A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/enzymology , NADP Transhydrogenases/metabolism , Rhodospirillum rubrum/enzymology , Amino Acid Sequence , Animals , Antibodies, Bacterial/pharmacology , Antibodies, Monoclonal/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Catalysis , Detergents/pharmacology , Hydrogen-Ion Concentration , Liposomes , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , NAD/analogs & derivatives , NAD/metabolism , NADP/metabolism , NADP Transhydrogenases/chemistry , NADP Transhydrogenases/immunology , Protein Binding , Protein Conformation , Protein Denaturation , Protein Multimerization , Protein Structure, Tertiary , Protons , Recombinant Fusion Proteins/metabolism , Trypsin/pharmacology
3.
Gene ; 212(1): 21-9, 1998 May 28.
Article in English | MEDLINE | ID: mdl-9661660

ABSTRACT

The 40-kDa antigen of M. tuberculosis, which is an alanine dehydrogenase, is a species-specific antigen that is potentially useful for strain identification. Large quantities of the purified protein are required for immunological, as well as for detailed biochemical and structural, characterization. The AlaDH gene was cloned by PCR from H37Rv (virulent) and H37Ra (partially attenuated) strains of M. tuberculosis, and their DNA sequence was determined. A host-vector system suitable for the production of sufficient quantities of the recombinant AlaDH antigen was developed. The AlaDH gene was expressed under the control of strong, transcriptional (bacteriophage pLpR) and translational (atpE) signals. High-level expression of soluble AlaDH was obtained using the recombinant E. coli K-12 strain CAG629 [pMSK12], which is deficient in Lon protease and the heat-shock response. A simple two-step procedure for the rapid purification of the recombinant protein was developed. The protein was purified to near homogeneity, and the purified AlaDH showed a specific enzyme activity comparable to the native protein isolated from M. tuberculosis. In addition, the product showed an expected amino acid sequence and reacted strongly to the 40-kDa (AlaDH)-specific mAb HBT-10. Furthermore, the epitope of the mAb HBT-10 was mapped to a 12-amino-acid region. Contrary to the published results, we show that the AlaDH and the PNT (pyridine nucleotide transhydrogenase) of M. tuberculosis do not share common epitopes reacting to the species-specific mAb HBT-10. The availability of highly purified AlaDH should now enable a detailed biochemical and structural characterization of this important enzyme of M. tuberculosis.


Subject(s)
Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/isolation & purification , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Alanine Dehydrogenase , Amino Acid Oxidoreductases/biosynthesis , Amino Acid Sequence , Antibodies, Monoclonal , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Antigens, Bacterial/isolation & purification , Base Sequence , Cross Reactions , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Genetic Vectors , Molecular Sequence Data , Mycobacterium tuberculosis/immunology , NADP Transhydrogenases/immunology , Plasmids/genetics , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
4.
J Appl Bacteriol ; 77(6): 639-43, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7822223

ABSTRACT

A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of Mycobacterium tuberculosis H37Rv with immobilized metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M(r) of 43,000 and a pI of approximately 6.0. The N-terminal amino acid sequence of TB43 was met-arg-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe-arg-val-ala- ile-thr-pro-ala. It showed 86% homology with the N-terminal end of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alanine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogenase of Myco. tuberculosis. It was also not recognized by MAb F29-29 which is known to react with a 43 kDa protein of Myco. tuberculosis complex. This protein exhibited strong Tsh activity. A similar 43 kDa protein showing Tsh activity was also isolated by IMAC from Myco. bovis CS. However, the pI of the protein was approximately 7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H37Ra. TB43 is a cell-associated pyridine nucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.


Subject(s)
Mycobacterium tuberculosis/enzymology , NADP Transhydrogenases/isolation & purification , Alanine Dehydrogenase , Amino Acid Oxidoreductases/analysis , Amino Acid Sequence , Antigens, Bacterial/isolation & purification , Blotting, Western , Chromatography, Affinity , Cross Reactions , Molecular Sequence Data , Mycobacterium tuberculosis/immunology , NADP Transhydrogenases/immunology , Sequence Analysis
5.
FEMS Microbiol Lett ; 110(2): 223-9, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-8349094

ABSTRACT

cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella. Monospecific antibodies were raised against the recombinant expression product. Using these antibodies, the parasite proteins were found to be localised in the refractile bodies. The derived amino-acid sequences were compared by computer using the SWISSPROT protein database. In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli. Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins. It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria.


Subject(s)
Carbohydrate Metabolism , Eimeria/metabolism , Monosaccharide Transport Proteins/metabolism , NADP Transhydrogenases/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Cloning, Molecular , DNA/genetics , DNA, Protozoan/genetics , Eimeria/genetics , Eimeria/immunology , Eimeria/ultrastructure , Molecular Sequence Data , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/immunology , NADP Transhydrogenases/genetics , NADP Transhydrogenases/immunology , Organelles/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid
6.
J Biol Chem ; 266(9): 5728-35, 1991 Mar 25.
Article in English | MEDLINE | ID: mdl-2005110

ABSTRACT

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.


Subject(s)
Mitochondria, Heart/enzymology , NADP Transhydrogenases/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cattle , Cell Membrane/enzymology , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Endopeptidase K , Enzyme-Linked Immunosorbent Assay , Immune Sera , Molecular Sequence Data , NADP Transhydrogenases/immunology , Protein Conformation , Serine Endopeptidases , Submitochondrial Particles/enzymology
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