Biomedical subjects

R W Ashton

Publications and source records attributed to R W Ashton.

7 recordsLinked to original sources

PubMed1997

Kinetics of competitive binding with application to thrombin complexes.

The kinetics of competitive binding is treated analytically, allowing the rate constants to be determined accurately from simple experiments. The method is especially suited to situations where traditional approximations and numerical integration fail, e.g., when the dissociation constants are small or when the concentration of one receptor cannot be measured accurately. The method is applied to the competitive binding of hirudin to thrombin and anhydrothrombin and found to be accurate to a few parts in ten million. The fitted rate constants show that anhydrothrombin binds hirudin more weakly than thrombin, with a 2.6-fold increase in its dissociation constant. The small relative difference in binding free energy (0.6 kcal/mol indicates that anhydrothrombin is structurally similar to thrombin.

Binding, Competitive↗

PubMed1995

Effects of temperature and glycerides on the enhancement of Agkistrodon piscivorus piscivorus phospholipase A2 activity by lysolecithin and palmitic acid.

The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature-dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer.

Agkistrodon↗

PubMed1995

Preparation and characterization of anhydrothrombin.

Anhydrothrombin, a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehydroalanine by promoting the beta-elimination of phenylmethylsulfonic acid from phenylmethylsulfonyl fluoride-inactivated thrombin under conditions in which the enzyme is unfolded. After the elimination reaction was quenched, the resulting anhydrothrombin was folded by diluting the denaturant, Gdn.HCl, to nondenaturing concentrations. Anhydrothrombin was purified by PAB affinity chromatography. Both native thrombin and anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by reverse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptide, as shown by amino acid analysis. This anhydrothrombin peptide was found to be 18.7 +/- 1.6 lower in mass units than the native peptide by electrospray mass spectrometry, in accord with the elimination of a water molecule. The anhydrothrombin preparation was monomeric, as determined by sedimentation equilibrium. Anhydrothrombin was used in a competitive titration of the complex of native thrombin with the leech saliva protein hirudin, a potent thrombin inhibitor, as measured by the recovery of thrombin amidolytic activity. This demonstrated that anhydrothrombin is capable of nativelike binding interactions with macromolecular ligands.

Animals↗

PubMed1995

Tetracyanoquinodimethane mediated glucose sensor based on a self-assembling alkanethiol/phospholipid bilayer.

An amperometric tetracyanoquinodimethane (TCNQ) mediated biosensor for glucose is described, based on a self-assembling alkanethiol/phospholipid bilayer laid down onto a gold surface. Gold was sputter deposited onto chromium coated silicon wafers to a thickness of 200 nm. A monolayer of alkanethiol was allowed to self-assemble from an ethanolic solution of dodecanethiol onto a freshly cleaned gold electrode in an overnight incubation. The monolayer was characterized by ellipsometric, impedance and cyclic voltammetry measurements (capacitance = 1.60 +/- 0.06 microF/cm2, and thickness 1.34 +/- 0.15 nm). A mixture of phospholipid liposomes containing free amino groups was placed on the monolayer and allowed to incubate overnight. The self-assembly of a phospholipid monolayer and allowed to incubate overnight. The onto the alkanethiol monolayer, resulted in the formation of a bilayer. The formation of bilayer was again characterized by impedance and cyclic voltammetry measurements (capacitance = 0.98 +/- 0.09 microF/cm2, and thickness = 1.85 +/- 0.22 nm). TCNQ has been incorporated into the liposomes before the formation of the bilayer. Glucose oxidase was cross-linked with the amino-groups of the phospholipids using bis [2-(sulphosuccinimiidooxicarbonyloxy) ethyl] sulphone. TCNQ which was incorporated in the bilayer acted as an efficient mediator to regenerate glucose oxidase. Cyclic voltammetry of the modified electrode and a response curve for the glucose sensor are reported.

Biosensing Techniques↗

PubMed1992

Thrombin hydrolysis of an N-terminal peptide from fibrinogen Lille: kinetic and NMR studies.

Fibrinogen Lille, a congenital dysfibrinogenemia, has been reported to arise from a mutation from Asp to Asn at position 7 of the A alpha chain of human fibrinogen, thereby reducing the thrombin-catalyzed rate of hydrolysis of the Arg(16)-Gly(17) peptide bond of this chain. Synthetic peptides of relevant portions of the wild-type and mutant A alpha chains were prepared, and the thrombin-catalyzed rates of hydrolysis of their Arg(16)-Gly(17) peptide bonds were determined. In addition, transferred NOE measurements were made to deduce their conformations, when complexed to bovine thrombin. The kinetics data showed little difference in the hydrolysis rates between the wild-type and mutant peptides, and the NMR data indicate no difference in the bound conformation of these two peptides. Therefore, electrostatic (or salt-bridge) interactions between Asp(7) and thrombin do not influence the bound conformations of these peptides. Asp(7) may interact with a remote residue of fibrinogen, not present in these synthetic peptides, or there may be additional mutations beyond A alpha (1-20) which have not been detected in fibrinogen Lille. Alternatively, when thrombin binds to fibrinogen at its secondary binding site, its primary (active) site may display different reactivities toward wild-type fibrinogen and fibrinogen Lille.

Afibrinogenemia↗

PubMed1989

Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site.

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).

Affinity Labels↗

PubMed1984

Enzymatic coupled assay procedures that employ high-performance liquid chromatography: the synthesis of orotidylate from ribose.

High-performance liquid chromatographic assay procedures have been designed to monitor the catalytic activities of ribokinase and phosphoribosyl alpha-1-pyrophosphate (PRibPP) synthetase. These methods are only of qualitative value, when crude protein extracts are to be examined, because of the presence of myokinase. However, the product of the PRibPP synthetase reaction, can be detected quantitatively even in crude protein extracts through the addition of two enzymes (orotate phosphoribosyltransferase and inorganic pyrophosphatase) that catalyze the conversion of PRibPP into a spectroscopically detectable nucleotide product (orotidylate).

Bacterial Proteins↗