An optical biosensor system for molecular interaction studies.
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Animals↗
Biomedical subjects
D Pollard-Knight
Publications and source records attributed to D Pollard-Knight.
Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase.
The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 x 10(5) molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-5.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3571 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe.
Blotting, Southern↗
Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence.
A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.
Alkaline Phosphatase↗
Immunoassays and nucleic acid detection with a biosensor based on surface plasmon resonance.
A technique based on surface plasmon resonance is described which can be used to detect changes of refractive index that occur when one partner of a molecular binding pair diffuses from solution to bind the other partner which is immobilised on a silver surface. Results for the molecular binding pairs; protein-antibody, hapten-antibody and DNA-DNA are described. Instrumentation necessary for implementation of the technique is detailed. Immunoassay of proteins and haptens is possible in less than one minute with a sensitivity of 10(-9) mol/l. Hybridisation of 10 fmoles of a 97 base target sequence on the 1 mm2 area of detection to an immobilised oligonucleotide probe can be detected in less than five minutes. Advantages of the technique include the ability to record the kinetics of binding reactions in "real time" and the lack of labels in this simple assay format. Methods of improving the sensitivity are discussed.
Animals↗
Kinetics of hexokinase D ('glucokinase') with inosine triphosphate as phosphate donor. Loss of kinetic co-operativity with respect to glucose.
When ATP, the normal phosphate donor for hexokinase D ('glucokinase'), is replaced by ITP, the positive co-operativity with respect to glucose disappears. This may be rationalized in relation to kinetic models for hexokinase D co-operativity, which assume that with the normal substrates the chemical reaction and subsequent release of products occur so rapidly that binding of substrates cannot approach equilibrium and is therefore not constrained by the thermodynamic requirement that the Hill coefficient for substrate binding cannot exceed the number of binding sites. ITP is a much poorer substrate than ATP, however: its Km value at high glucose concentrations is 24 times the value for ATP, whereas the value of the limiting rate V is decreased about 8-fold. Consequently it is no longer possible for the ternary complex to be converted into products rapidly enough to generate kinetic co-operativity. The negative co-operativity with respect to glucose observed in 2H2O with ATP as phosphate donor also disappears when ITP is used instead of ATP.
Animals↗
Effect of glycerol on glucokinase activity: loss of cooperative behavior with respect to glucose.
Glucose phosphorylation catalyzed by rat liver glucokinase measured at saturating concentrations of MgATP2- shows a cooperative response with respect to glucose in the concentration range 0.25-5 mM with a Hill coefficient of 1.6. In this range of glucose concentrations, the degree of cooperativity was dependent on the presence of glycerol in the assay mixture, and it decreased progressively and disappeared completely as the glycerol concentration reached about 20% (v/v) glycerol. If attention was confined to concentrations above 5 mM, no cooperativity could be detected either in the absence or in the presence of glycerol. The limiting velocity of the glucokinase reaction (measured at saturating concentrations of glucose and MgATP2-), and the half-saturation concentration for glucose and MgATP2- were all decreased by about 50-60% as the glycerol concentration was raised from zero to 30% (v/v). The presence of glycerol had no effect on the qualitative inhibition patterns of MgADP2-, glucose 6-phosphate, or N-acetylglucosamine, and only slight effects on the quantitative half-saturation values and inhibition constants. All of these effects caused by glycerol were fully reversible by decreasing the concentration of glycerol by dilution. Simulation studies based on the "mnemonical" model of glucokinase action proposed earlier [A. C. Storer and A. Cornish-Bowden (1977) Biochem. J. 165, 61-69] show that the effects of glycerol on glucokinase-catalyzed glucose phosphorylation can simply be explained assuming the glycerol favors the existence of the conformation of the enzyme with a higher affinity for glucose and thus supports the model.
Adenosine Triphosphate↗
Solvent isotope effects on the hexokinase D reaction: evidence for the mnemonical interpretation of the kinetic co-operativity.
Hexokinase D ('glucokinase') from rat liver displays kinetic co-operativity with respect to glucose when studied in 1H2O, with a Hill coefficient of about 1.4 when the other substrate, MgATP, is present at a concentration of 4.3 mM. In 2H2O, however, this becomes negative co-operativity, with a Hill coefficient of about 0.6 under corresponding conditions. At high glucose concentrations there is a small normal isotope effect, but at low glucose concentrations there is a large inverse isotope effect. Graphical analysis shows that these results are consistent with the 'mnemonical' model for kinetic co-operativity, in which there are two forms of free enzyme with different affinities for glucose, if the principal effect of changing the solvent is to alter the relative magnitudes of the on rate constants for binding of glucose to the two forms of free enzyme.
Adenosine Triphosphate↗
Solvent isotope effects on the glucokinase reaction. Negative co-operativity and a large inverse isotope effect in 2H2O.
The solvent isotope effects on the reaction catalysed by rat-liver glucokinase have been studied. At low concentrations of glucose and high concentrations of MgATP2- there is an inverse solvent isotope effect of 3.5. At high glucose concentrations there is a normal solvent isotope effect of 1.3. In 1H2O there is positive co-operativity with respect to glucose [ Storer , A.C. and Cornish - Bowden , A. (1976) Biochem. J. 159, 7-14], but this is changed to negative co-operativity in 2H2O. The half-saturation points for both glucose and MgATP2- are decreased in 2H2O compared with those in 1H2O. Explanations of these effects in terms of the mnemonical model proposed by Storer and Cornish - Bowden [Biochem. J. 65, 61-69 (1977)] were considered in computer simulation. Two interpretations could account for the results, either a decrease in the rate of interconversion of the two forms of free enzyme postulated in the model, or an increase in the affinity for glucose of the enzyme form with the lower affinity in 1H2O. The results of a proton-inventory analysis were consistent with either of these interpretations. The solvent isotope effects thus provide additional evidence for the mnemonical model as an explanation of glucokinase co-operativity.
Adenosine Triphosphate↗
Mechanism of liver glucokinase.
Glucokinase is the enzyme primarily responsible for the phosphorylation of glucose in the livers of mammals and other vertebrates. It differs from the other hexokinases in being insensitive to inhibition by glucose 6-phosphate and in responding co-operatively to changes in the glucose concentration in the physiological range. These properties accord well with the presumed function of glucose phosphorylation in the liver as a means of controlling the blood-glucose concentration. Glucokinase has the unusual property for a co-operative enzyme of being a monomeric enzyme with a single active site. The co-operativity consequently requires a purely kinetic explanation and cannot be explained by analogy with subunit interactions in proteins that display co-operativity in equilibrium binding. The behaviour is consistent with a 'mnemonical' type of mechanism, i.e. one in which the co-operativity derives from the occurrence of two interconvertible forms of free enzyme that are not at equilibrium in the steady state. As co-operativity is observed only with glucose and not with the other substrate, MgATP2-, a corollary of this interpretation is that glucose must bind predominantly or exclusively before MgATP2-. This order of binding is supported by isotope-exchange measurements, though the alternative order also appears to be possible as a minor route of reaction. Stereochemical investigations reveal that glucokinase resembles other hexokinases in that the form of MgATP2- that reacts with the enzyme is the beta gamma-bidentate complex with the lambda-screw sense, and that the reaction proceeds with inversion of configuration at phosphorus.
Acetylglucosamine↗
The stereochemical course of phosphoryl transfer catalysed by glucokinase.
Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an 'in-line' mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.
Adenosine Triphosphate↗