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Biomedical subjects

A R Walmsley

Publications and source records attributed to A R Walmsley.

12 recordsLinked to original sources
PubMed1992

Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.

A kinetic and thermodynamic analysis has been carried out on the conformational transitions of the periplasmic C4-dicarboxylate binding protein (DctP) from the photosynthetic bacterium Rhodobacter capsulatus. This protein is distinct from other periplasmic binding proteins characterized to date in that the transition between the putative closed-unliganded (BP1) and open-unliganded (BP2) conformations is slow compared to the rate of ligand binding [Walmsley, A. R., Shaw, J. G., & Kelly, D. J. (1992) J. Biol. Chem. 276, 8064-8072]. Using stopped-flow fluorescence techniques, we have probed the conformational dynamics of the closed to open transition of DctP in the absence and presence of ligand. Both the forward rate constant for the BP1 to BP2 interconversion (k1) and the fumarate dissociation rate constant (k-3) were found to increase in a biphasic manner between pH 5 and pH 11. The data were fitted to a two-pKa function which gave pKa values of 10.3 and 5.4 for the BP1 to BP2 interconversion and 8.9 and 4.5 for the closed-liganded (BP3L) to open-liganded (BP2L) transition. An increase in ionic strength at constant pH resulted in a hyperbolic increase in both k1 and k-3 to maximal rates that were similar in each case to the values obtained in pH variation experiments. Measurement of the temperature dependencies of k1 and k-3 also gave similar activation energies. Gibbs free energy, enthalpy, and entropy changes were determined for the open to closed transitions of DctP in both the presence and absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗
PubMed1992

Kinetic studies of the polygalacturonase enzyme from Colletotrichum lindemuthianum.

The intrinsic protein fluorescence of the polygalacturonase from Colletotrichium lindemuthianum was exploited in stopped-flow experiments aimed at elucidating the kinetic mechanism for this enzyme. Binding of the polymeric substrate polygalacturonic acid (PGA) essentially produced a triphasic fluorescence profile. There was an initial rapid quench in fluorescence, consistent with the rapid formation of the enzyme-substrate complex, with an equilibrium constant of about 8 x 10(-4)% (w/v) PGA (about 0.27 microM). There then followed a near-constant fluorescence phase, attributable to turnover of the enzyme-substrate complex as a steady-state intermediate. As the concentration of the steady-state intermediate became depleted, towards the end of the reaction, there was a partial return of the fluorescence intensity. This phase is attributed to a final, single turnover of the enzyme at the end of the reaction. The fluorescence intensity does not return to its original level due to product remaining bound at the end of the reaction.

Kinetics↗
PubMed1992

The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.

The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.

Bacterial Proteins↗
PubMed1990

The calcium ion dependence of scallop myosin ATPase activity.

The ATPase activity of scallop (Pecten maximus) striated adductor myosin and heavy meromyosin (HMM) have been investigated as a function of [Ca2+] using formycin triphosphate (FTP) as a fluorescent ATP analogue. The FTPase activity of the regulated fraction of these preparations was activated steeply over the range of 0.1 to 1 microM [Ca2+], implying the existence of a form of cooperativity that is intrinsic to the myosin heads. In addition to the previously characterised heterogeneity with respect to an unregulated fraction, the regulated fraction of HMM was resolved into two populations whose activities showed a slightly different dependency on [Ca2+]. This was revealed unambiguously at intermediate levels of activation where, in some experiments, the product release rate constants differed for the two populations by more than fivefold. At maximum relaxation or maximum activation, these rate constants differed by two- to three-fold and were not clearly resolved by the multiexponential fitting procedure. The populations might arise as a consequence of isoenzymes, modification during preparation or slowly interconverting conformers; Ca2+ binding itself being a rapid equilibrium process in both populations. FTP turnover by myosin could not be analysed in such detail because of the technical problems of measuring the fluorescence of a suspension of filaments, but the rates of the elementary steps appeared similar to those of HMM. The fraction of unregulated molecules in myosin preparations was comparable to that of HMM indicating that if it is a consequence of preparative damage, the modification must occur prior to tryptic digestion.

Animals↗
PubMed1989

Logarithmic timebase for stopped-flow data acquisition and analysis.

A method for capturing stopped-flow and other rapid reaction records in logarithmic time, using a microcomputer, is described. Apart from the ability to record processes over several decades in time in a single experiment, the method shows distinct advantages in subsequent nonlinear regression analysis of multiexponential processes. The method is illustrated by a study of the binding of NADPH to dihydrofolate reductase and the reaction between formycin triphosphate and heavy meromyosin.

Animals↗
PubMed1987

A single half-turnover of the glucose carrier of the human erythrocyte.

Single half-turnovers of the glucose carrier of the human erythrocyte have been measured by recruiting carriers to the outward-facing conformation by (a) pre-exposing cells to extracellular maltose, or (b) pre-warming cells to 38 degrees C, before addition of D-[14C]glucose at 0 degrees C. Based on these experiments estimates of the number of glucose carriers per red cell range from 124,000 to 190,000.

Blood Glucose↗
PubMed1987

Comparison of the kinetics and thermodynamics of the carrier systems for glucose and leucine in human red blood cells.

Kinetic data for the transport of glucose and leucine in human red blood cells are fitted to the conventional carrier model and the thermodynamics of the two carrier mechanisms are compared. In the absence of the carried molecule both carriers exist mainly in the inward-facing conformation at low temperatures and the outward-facing conformation at physiological or supra-physiological temperatures, this finding reflecting the strongly endothermic process involved in changing from the inward- to outward-facing forms. Reorientations from inward- to outward-conformations also involve substantial increases in entropy for both carriers. In contrast, substrate binding to the glucose carrier involves little change in enthalpy and an increase in entropy, while leucine binding is strongly exothermic and associated with a decrease in entropy. Application of transition state theory to glucose carrier kinetics reveals that the entropy of formation of the transition state of the carrier is much greater than that for the transition state of the carrier-glucose complex.

Carrier Proteins↗
PubMed1986

The kinetics of glucose transport in human red blood cells.

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.

Biological Transport, Active↗
PubMed1985

A quenched-flow technique for the measurement of glucose influx into human red blood cells.

A quenched-flow apparatus is described and applied to measurements of the hydrolysis of 2,4-dinitrophenyl acetate by sodium hydroxide and the entry of D-[U-14C]glucose into human red blood cells at 37 degrees C. Glucose influx into red cells was a saturable process obeying Michaelis-Menten kinetics with a Km for glucose of 6.6 +/- 0.61 mM and a maximum rate for glucose entry under "zero trans" conditions of 20.7 +/- 0.76 mmol (L cell water)-1 s-1. The technique used requires only readily available laboratory equipment and should be easily adaptable to the study of other rapid transport processes.

Biological Transport↗
PubMed1985

Multifit: a flexible non-linear least squares regression program in BASIC.

MULTIFIT, a program in BASIC for implementation on microcomputers, has been developed for non-linear least squares regression fitting of enzyme kinetic, pharmacokinetic and other data to specific models. The program contains a simple procedure for insertion of model equations with up to five parameters (to be fitted) up to 3 independent variables and 1 dependent variable, and can be used to generate a family of programs with pre-set model functions.

Computers↗