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Biomedical subjects

T Atkinson

Publications and source records attributed to T Atkinson.

At least 163 records · Page 9Linked to original sources
PubMed1983

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus.

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.

Chromatography, Affinity↗
PubMed1983

Properties of thyP3-containing plasmids in Bacillus subtilis.

A series of hybrid plasmid molecules which contain both antibiotic resistance genes and the thyP3 gene of the Bacillus subtilis bacteriophage phi 3T have been constructed. Monomeric or restriction enzyme-cleaved plasmid DNA is capable of transforming competent cells to thymine prototrophy only. However, multimeric plasmid DNA can transform competent cells to both thymine prototrophy and antibiotic resistance. Cells which have been transformed to thymine prototrophy only do not contain extrachromosomal plasmid DNA but instead contain the thyP3 gene integrated into the host chromosome; the antibiotic resistance genes, however, do not become integrated into the chromosome. Although the thyP3-containing plasmids have extensive DNA sequence homology with the B. subtilis chromosome, they can be stably maintained, extrachromosomally, even in recE4+ hosts, in complex broth, and in the absence of antibiotics.

Acetyltransferases↗
PubMed1983

The R-factors of multiple antibiotic resistant faecal coliforms isolated from a domestic dog.

The antibiotic resistant faecal flora of a domestic dog suffering from an acute enteric infection was examined. The flora exhibited overall resistance to a wide variety of antibiotics. However, following restoration of the animal to normal health, overall resistance to ampicillin (Ap), tetracycline (Tc), chloramphenicol (Cm) and streptomycin Sm was lost, although low numbers of bacteria resistant to these four antimicrobial agents could still be isolated up to one year later. A total of 11 strains were purified for further study. All 11 were positively identified as Escherichia coli and shown to be resistant to various combinations of the above antibiotics, and additionally to kanamycin (Km). Each strain harboured from one to five plasmids, although only four proved capable of transferring antibiotic resistance to Escherichia coli K-12. One of the strains was found to harbour two conjugal plasmids pNJ101 (60 Md) and pNJ102 (133 Md) which coded for resistance to Cm, Tc, Ap and Cm, Tc, Km respectively. A third plasmid pNJ103 (29 Md) remains cryptic. The possession of the two plasmids pNJ101 and pNJ102 appears to be an unstable situation as variants arose harbouring one or other of the plasmids.

Animals↗
PubMed1983

Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida.

The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp. strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322. The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning. The expression of carboxypeptidase in E. coli was 100-fold lower than in the Pseudomonas sp. strain. When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E. coli was observed. High expression (up to 5% soluble protein) was obtained in E. coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153. The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector. Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E. coli.

Carboxypeptidases↗
PubMed1983

Production of a fibrinolytic enzyme from cultures of guinea pig keratocytes grown on microcarriers.

Microcarrier technology was investigated as a means of producing a fibrinolytic enzyme, for the treatment of occlusive thrombotic vascular disorders, in sufficient quantity to carry out an evaluation of its clinical potential and the economics of large scale production. Preliminary results showed that the cells grew well and produced significant quantities of the enzyme at high (10 g/1) microcarrier concentrations.

Animals↗
PubMed1982

The rapid purification of 3-hydroxybutyrate dehydrogenase and malate dehydrogenase on triazine dye affinity matrices.

3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were purified to homogeneity on a large scale involving only two sequential affinity-chromatography steps on two triazine dye-Sepharose matrices. Recoveries of both enzymes were in excess of 60%. Malate dehydrogenase could also be purified by a combination of triazine dye affinity chromatography and gel filtration on Ultrogel AcA-44, but this offered no significant advantage over the purely affinity procedure.

Chromatography, Affinity↗
PubMed1982

The interaction of tryptophanyl-tRNA synthetase with the triazine dye Brown MX-5BR.

Brown MX-5BR specifically and irreversibly inactivates tryptophanyl-tRNA synthetase from Bacillus stearothermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product inorganic pyrophosphate and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme was measured to be 6.7 X 10(-6) M. The Km values of the two substrates tryptophan and MgATP were found to be 1 x 10(-5) M and 5 x 10(-5) M respectively. The aminated dye is a competitive inhibitor of tryptophanyl-tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2 x 10(-4) M against both substrates. The use of this dye as an active-site-directed affinity label is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Affinity labelling of enzymes with triazine dyes. Isolation of a peptide in the catalytic domain of horse-liver alcohol dehydrogenase using Procion blue MX-R as a structural probe.

Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion blue MX-R, a dichlorotriazinyl structural analogue of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37 degrees C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 microM. Methoxylated Procion blue MX-R does not inactivate the enzyme. The inactivation of horse liver alcohol dehydrogenase by Procion blue MX-R is competitively inhibited by the pyridine nucleotides NAD+ and NADH. Quantitatively inhibited horse liver alcohol dehydrogenase contains 1 mol dye/mol subunit of Mr 40 000. Chymotryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a single blue peptide which on sequencing and analysis yields an amino acid sequence of: (formula; see text) with the affinity label, Procion blue MX-R, unambiguously identified as being attached to the thiol side chain of Cys-174 in the catalytic domain of the enzyme. The specific active-site-directed reaction of Procion blue MX-R with horse liver alcohol dehydrogenase is interpreted in terms of the known crystallographic structure of the enzyme.

Affinity Labels↗
PubMed1981

The effect of lactation length on the circulating concentrations of progesterone and oestradiol in the early weaned sow.

Eighteen crossbred multiparous sows were allocated at random to one of two lactation lengths: 42 or 10 days. All sows were mated at the oestrus after weaning and from mating until day 26 post coitum they were bled every second day. Progesterone and oestradiol concentration in the plasma was determined by radioimmunoassay. Progesterone increased more rapidly between days 4 and 10 post coitum in early weaned sows and the oestradiol surge at mating was abnormally extended for the same group.

Journal Article↗
PubMed1980

Characterization of Bacillus stearothermophilus plasmid pab124 and construction of deletion variants.

A restriction endonuclease cleavage map of the tetracycline resistance plasmid pAB124, originally isolated from Bacillus stearothermophilus, was constructed using ten enzymes. Tetracycline resistance was associated with a 1 x 95 megadalton (Md) region of pAB124 lying between two EcoRI sites, and this region was circularized to produce a viable tetracycline resistance plasmid (pAB224), with two EcoRI fragments of pAB124 deleted amounting to 0 x 95 Md. A second plasmid (pAB524) with one EcoRI fragment (0 x 6 Md) of pAB124 deleted was also constructed. Restriction endonuclease cleavage maps of pAB224 and pAB524 were constructed.

Cloning, Molecular↗
PubMed1979

The binding of aminoacyl-tRNA synthetases to triazine dye conjugates.

The binding of thirteen aminoacyl-tRNA synthetases to thirty two immobilised procion dyes has been investigated. Most dyes bind one or more enzymes. The amino acid substrates are not normally potent eluants, with the notable exception of tryptophan eluting tryptophanyl-tRNA synthetase from Brown MX-5BR. Phosphate is frequently extremely effective, much more than expected by simple considerations of ionic strength, indicating that many of the dyes are able to mimic the phosphate groups of the phosphodiester backbone of the nucleic acid. Procedures for the purification of methionyl-, tryptophanyl- and tyrosyl-tRNA synthetases are presented and compared to the conventional purifications of these enzymes. The results indicate the general applicability of these dye columns to the purification of most enzymes of of nucleic acid metabolism and the necessity of investigating as many different dyes as possible for any individual enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1979

The optimal amino acid supplementation of barley for the growing pig. 1. Response of nitrogen metabolism to progressive supplementation.

1. In five experiments, involving 142 female pigs weighing on average 33 kg, estimates were made of the amounts of essential amino acids which minimized urinary N excretion when diets with barley as the only source of protein were given at the rate of 120 g/kg0.75 per d. 2. With additions of lysine (4.0 g/kg diet) and threonine, (1.2 g/kg diet) to barley urinary N excretion decreased from 0.91 to 0.36 g/kg0.75 per d, corresponding to an increase in biological value (bv) from 0.51 to 0.86. 3. With these additions of lysine and threonine, there were no responses to additions of tryptophan, methionine or isoleucine, or to further additions of lysine or threonine, but addition of histidine significantly reduced N excretion. 4. No optimal addition of histidine could be determined; the mean rate of N excretion after addition of histidine (not less than 0.3 g/kg diet) was 0.27 g/kg0.75 per d, corresponding to a BV of 0.93. 5. There was a variation between pigs from different litters in their responses to added histidine. Those with low rates of N excretion on the unsupplemented diet did not respond to additions of histidine, but those with high rates did. 6. It is concluded that additions of only three amino acids can greatly improve the nutritive value of barley protein for the growing pig and that the amino acid composition of the supplemented protein closely approaches the ideal; it is also similar to whole-body tissue protein.

Amino Acids, Essential↗