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Biomedical subjects

M J Danson

Publications and source records attributed to M J Danson.

At least 55 records · Page 3Linked to original sources
PubMed1992

The enzymology of archaebacterial pathways of central metabolism.

From a comparison of the pathways of central metabolism in the archaebacteria, eubacteria and eukaryotes, it is clear that the basic pathways were established before the divergence of the three kingdoms, but that the notable differences may provide important clues to their evolution. From these comparisons, enzymes found in all evolutionary groups have been chosen for detailed structural studies; given the range of extreme phenotypes found within the archaebacteria, these studies will be crucial to our understanding of the structural basis for protein stability and how such features may be engineered into a protein of choice.

Archaea↗
PubMed1991

Crystallization and preliminary crystallographic study of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum.

Single crystals of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group P6122 or P6522, with unit cell dimensions a = b = 121.9 angstrom, c = 229.6 angstrom and with two molecules in the asymmetric unit.

Bacterial Proteins↗
PubMed1991

Expression and purification of plasmid-encoded Thermoplasma acidophilum citrate synthase from Escherichia coli.

The citrate synthase gene from the thermophilic archaebacterium Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight. Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E. coli ribosome. A rapid purification of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

Base Sequence↗
PubMed1990

Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilium. Cloning and sequencing of the gene.

The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum. We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. The similarity is less than 30% between the archaebacterial and non-archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms. The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase. This is the first reported sequence of citrate synthase from the archaebacteria.

Amino Acid Sequence↗
PubMed1990

Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei.

In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661-665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane. The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T. brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.

Animals↗
PubMed1989

Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/- 3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E.

Amino Acid Sequence↗
PubMed1989

Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure.

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

Bacterial Proteins↗
PubMed1989

Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices.

The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids. The alignment studies have been performed as the first step in a project aimed at homology modelling E. coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes. The effects of several important variables in producing realistic alignments have been investigated. The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E. coli proteins was explored. The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated. The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.

Algorithms↗
PubMed1988

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.

Animals↗
PubMed1988

A new spectrophotometric assay for citrate synthase and its use to assess the inhibitory effects of palmitoyl thioesters.

We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.

Acyl Coenzyme A↗
PubMed1987

Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location.

Dihydrolipoamide dehydrogenase has been discovered in the bloodstream form of the eukaryotic African parasite, Trypanosoma brucei. The enzyme catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+ and exhibited a hyperbolic dependence of catalytic activity on the concentrations of both dihydrolipoamide and NAD+. Chemical modification with the tervalent arsenical reagent p-aminophenyldichloroarsine indicates the involvement in catalysis of a reversibly reducible disulphide bond. Plasma-membrane sheets were purified from T. brucei, and it was shown that virtually all the dihydrolipoamide dehydrogenase remained closely associated with this membrane preparation. T. brucei apparently lacks the 2-oxoacid dehydrogenase multienzyme complexes of which dihydrolipoamide dehydrogenase is usually an integral component. In the context of this absence, the possible function of trypanosomal dihydrolipoamide dehydrogenase is discussed, with particular reference to its cellular location in the plasma membrane.

Animals↗
PubMed1986

A comparison of ELISA screening methods for the production of monoclonal antibodies against soluble protein antigens.

A library of monoclonal antibodies against pig heart citrate synthase has been raised. Twelve solid-phase immunoassay systems, employing different methods of antigen immobilization, have been compared for their ability to detect the various members of this library. It was found that a sandwich immunoassay, in which the citrate synthase antigen was immobilized on the solid support via a polyclonal antibody preparation, was the only system capable of detecting all the monoclonal antibodies tested. It is suggested that such a sandwich assay should be used in preference to direct assays for the initial screening of monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1985

Citrate synthase: an immunochemical investigation of interspecies diversity.

Rabbit antibodies have been raised to pig heart citrate synthase. Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity. From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart greater than pigeon breast greater than Bacillus megaterium greater than Escherichia coli. These findings are discussed in terms of the known interspecies diversity of the enzyme.

Animals↗