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Biomedical subjects

C R Lowe

Publications and source records attributed to C R Lowe.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterisation of an NAD(+)-dependent secondary alcohol dehydrogenase from Pseudomonas maltophilia MB11L.

A constitutive NAD(+)-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose. Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols. Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5. The Km values for propan-2-ol, NAD+ and acetone were 87, 413 and 143 microM respectively. The enzyme is a tetramer with subunit Mr of approximately 44,000. It has an isoelectric point of 4.75, and was inhibited by chelating agents, thiol reagents and certain metal ions.

Alcohol Dehydrogenase↗
PubMed1992

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.

Chromatography, Affinity↗
PubMed1992

Design of novel affinity adsorbents for the purification of trypsin-like proteases.

A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de novo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.

Benzamidines↗
PubMed1992

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.

Binding Sites↗
PubMed1992

A single mode fibre-optic evanescent wave biosensor.

This paper reports experimental developments in the construction and operation of a single-mode fibre-optic evanescent wave biosensor using an exposed core silica single-mode fibre embedded in a silica block. The device was able to monitor the concentration of a blue dye, Procion Blue MX-G, in overlayers of various refractive indices. The practicality of such a biosensor has been demonstrated with a colorimetric enzyme assay system. Penicillin G in the 0-0.4 mM concentration range was monitored at 633 nm by the decoloration of the starch-iodine reagent when Bacillus cereus penicillinase was immobilized over the exposed core of the monomode fibre.

Biosensing Techniques↗
PubMed1992

Identification of a cocaine esterase in a strain of Pseudomonas maltophilia.

A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer. The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively. Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1. The cocaine esterase of P. maltophilia MB11L showed no activity with atropine, despite the structural similarity of cocaine and atropine.

Benzoates↗
PubMed1991

Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10.

The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.

Alcohol Oxidoreductases↗
PubMed1991

Monosized adsorbents for high-performance affinity chromatography. Application to the purification of calf intestinal alkaline phosphatase and human urine urokinase.

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphatase and human urine urokinase from crude extracts. This study demonstrates that the combination of specifically designed affinity ligands with monosized support materials is a powerful approach to the resolution of proteins by high-performance affinity chromatography.

Adsorption↗
PubMed1990

Biosensors.

This review introduces biosensors as analytical devices that respond selectively to analytes in appropriate samples and convert their concentrations into electrical signals via a combination of a biological recognition system and a suitable transducer. The last decade has seen dramatic advances in the design of sensor configurations, the marriage of biological systems with modern monolithic silicon and optical technologies, the development of effective electron-exchange systems and the introduction of direct immunosensors.

Animals↗
PubMed1990

Design and applications of biomimetic anthraquinone dyes. III. Anthraquinone-immobilised C.I. reactive blue 2 analogues and their interaction with horse liver alcohol dehydrogenase and other adenine nucleotide-binding proteins.

C.I. Reactive Blue 2 analogues were bonded onto an agarose support matrix by a novel method which entailed immobilisation by the anthraquinone ring 1-amino group as opposed to the usual triazine ring coupling methods. Dyes with spacer arms attached to the anthraquinone ring 1-amino group were synthesised by reacting methoxytriazine analogues of C.I. Reactive Blue 2 with chloroacetyl chloride and ethylenediamine. Unlike the blue parent dyes, all C.I. Reactive Blue 2 analogues with derivatised anthraquinone ring 1-amino groups were of a characteristic red colour. This change of chromaticity was entirely expected since the anthraquinone ring 1-amino group is an important component of the C.I. Reactive Blue 2 chromophore. Chromatographic studies indicated that, in comparison to adsorbents comprising triazine ring-immobilised dyes, adsorbents formed from C.I. Reactive Blue 2 analogues immobilised by the anthraquinone ring were better suited to the isolation of horse liver alcohol dehydrogenase and other adenine nucleotide-requiring enzymes. Similarities between C.I. Reactive Blue 2 analogues immobilised by the anthraquinone ring and N6-(6-aminohexyl)adenine nucleotide derivatives could be identified which may account for these observations. These studies confirm that highly effective affinity ligands based on synthetic textile dyes can be designed in a rational manner.

Adenine Nucleotides↗
PubMed1990

New developments in affinity chromatography.

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.

Biotechnology↗
PubMed1990

Microbial degradation of the morphine alkaloids: identification of morphine as an intermediate in the metabolism of morphine by Pseudomonas putida M10.

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively. This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the beta-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.

Biodegradation, Environmental↗
PubMed1989

Biosensors.

Biosensors are analytical devices that respond selectively to analytes in an appropriate sample and convert their concentration into an electrical signal via a combination of a biological recognition system and an electrochemical, optical or other transducer. Such devices will find application in medicine, agriculture, environmental monitoring and the bioprocessing industries. The last few years have seen great advances in the design of sensor architectures, the marriage of biological systems with monolithic silicon and optical technologies, the development of effective electron-transfer systems and the configuration of direct immunosensors. Recent progress in these areas has already led to the introduction of new-generation biosensors into the competitive diagnostics market place.

Biosensing Techniques↗
PubMed1989

Design and applications of biomimetic anthraquinone dyes. Purification of calf intestinal alkaline phosphatase with immobilised terminal ring analogues of C.I. reactive blue 2.

A 330-fold one-step purification of alkaline phosphatase from a crude calf intestinal extract has been achieved using specific elution with inorganic phosphate (5 mM) from a purpose designed adsorbent comprising a terminal ring phosphonate analogue of C.I. Reactive Blue 2 coupled to Sepharose CL-6B-200. The resulting alkaline phosphatase preparation displayed a specific activity in excess of 1000 U/mg and was of equivalent purity to commercial "high purity" preparations as deduced by sodium dodecyl sulphate polyacrylamide gel electrophoresis and specific activity comparisons.

Alkaline Phosphatase↗
PubMed1987

Introduction to the principles and applications of biosensors.

A biosensor is an analytical device that responds to an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and an electrochemical transducer. As a result of recent scientific and technological progress, such devices are likely to play an increasingly important role in generating analytical information in all sectors of human endeavour, from medicine to the military. In particular, biosensors will form the basis of cheap, simple devices for acquiring chemical information, bringing sophisticated analytical capabilities to the non-specialist and general public alike. The market opportunities for the rapid exploitation of novel developments in this sector are substantial. Biosensor research is also likely to have a significant impact on the development of modern electronics.

Biotechnology↗