Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases.

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states.

A simple tool to explore the distance distribution of correlated mutations in proteins.

The analysis of correlated mutations in protein sequence alignments is of considerable interest, since it may provide useful energetic and even structural information (ideally, residue contacts). However, a number of recent experimental studies support the existence of long-distance communication in proteins, a fact that may lead to correlation between distant residues. We introduce in this work a simple statistical procedure to describe the relation structure--alignments on the basis of the residue--residue distance dependence of the number of residue couples over given thresholds of a correlation measure (such as a covariance value).

Energetic evidence for formation of a pH-dependent hydrophobic cluster in the denatured state of Thermus thermophilus ribonuclease H.

NMR studies on the denatured states of proteins indicate that residual structure often resides predominantly in hydrophobic clusters. Such hydrophobic cluster formation implies burial of apolar surface and, consequently, is expected to cause a decrease in heat capacity. We report here that, in the case of ribonuclease H from the thermophile Thermus thermophilus, a sharp decrease in denatured-state heat capacity occurs at about pH 3.

Energetics of heparin binding to human acidic fibroblast growth factor.

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible.

Myo-inositol hexasulphate and low molecular weight heparin binding to human acidic fibroblast growth factor: a calorimetric and FTIR study.

The interaction of an amino-terminal-truncated 139 amino-acids form of human acidic fibroblast growth factor with myo-inositol hexasulphate and low molecular weight (3500 g mol(-1)) heparin has been studied by isothermal titration calorimetry, differential scanning calorimetry and Fourier transform infrared spectroscopy. A slightly higher affinity for the monosaccharide has been measured. The binding of the ligands causes an increase of 13--15 degrees C in the melting temperature of the free protein (45 degrees C).

Energetics of myo-inositol hexasulfate binding to human acidic fibroblast growth factor effect of ionic strength and temperature.

The binding of myo-inositol hexasulfate to an N-terminal truncated 132-amino-acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible.

Dissecting the energetics of the apoflavodoxin-FMN complex.

Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule.

Measurement of biochemical affinities with a Gill titration calorimeter.

A Gill titration calorimeter is evaluated as an instrument to determine in one experiment the equilibrium constant and the enthalpy change of a biochemical reaction. The dimensionless parameter kc (the product of the association equilibrium constant and the concentration of the reagent to be titrated; Wiseman et al., Anal.

Analogous activation of bovine liver glycogen phosphorylase by AMP and IMP.

The mechanism of activation of glycogen phosphorylase is incompletely understood, although adenosine and inosine nucleotides are known to be important allosteric activators. In this study the activation of glycogen phosphorylases a and b from bovine liver by adenosine 5'-monophosphate (AMP) and inosine 5'-monophosphate (IMP) has been investigated and the results compared with the activation of the muscle isozyme by the same nucleotides. Enzyme activity was determined by spectrophotometric measurement of inorganic phosphate produced in the phosphorylase-catalysed reaction of glycogen synthesis.

Maximum entropy, analysis of kinetic processes involving chemical and folding-unfolding changes in proteins.

We show that numerical inversion of the Laplace transform by using the maximum entropy method can be successfully applied to the analysis of complex kinetic processes involving chemical and folding-unfolding changes in proteins. First, we present analyses of simulated data which support that: (i) the maximum entropy calculation of rate distributions, combined with Monte Carlo analyses of the associated uncertainties, yields results consistent with the information actually supplied by the data, thus preventing their over-interpretation; (ii) maximum entropy analysis may be used to extract discrete rates (corresponding to individual exponential contributions), as well as broad rate distributions (provided, of course, that the adequate information is supplied by the data). We further illustrate the applicability of the maximum entropy analysis with experimental data corresponding to two nontrivial model processes: (a) the kinetics of chemical modification of sulfhydryl groups in glycogen synthase by reaction with Ellman's reagent; (b) the kinetics of folding of ribonuclease a under strongly folding conditions, as monitored by fluorescence and optical absorption.

Molecular studies of glycogen phosphorylase b from bovine liver.

Two active isoforms of bovine liver phosphorylase with distinct subunit composition have previously been purified (Cámara Artigas, A., Barón, C. and Parody-Morreale, A.

Purification of two forms of bovine liver glycogen phosphorylase b with distinct subunit composition.

The procedures for the purification of two forms of bovine liver glycogen phosphorylase b are described. Both forms showed a single band in nondenaturing gel electrophoresis. Gel electrophoresis in the presence of sodium dodecyl sulfate produced a single-band pattern for one of the enzyme forms (phosphorylase b1) and a triple-band pattern for the other (phosphorylase b3).

Differential scanning calorimetry of lobster haemocyanin.

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer.

Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins.

Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity.

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen.

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J.

A differential scanning calorimeter for ice nucleation distribution studies--application to bacterial nucleators.

A differential scanning calorimeter has been developed for the automatic detection and measurement of dropwise freezing within a sample of 100-200 water drops. A typical drop size of 1 microliter is employed. The sample is distributed on flat, square (4-cm) thermoelectric sensors and the temperature is scanned downward by conductive cooling to a liquid nitrogen bath.

A twin titration microcalorimeter for the study of biochemical reactions.

A small-volume (200 microliter) titration calorimeter of high sensitivity (1 mu cal ) has been developed for the purpose of studying biochemical reactions where the amounts of material are limited to a few nanomoles. High sensitivity is achieved by calorimetric twining , use of glass cells, elimination of vapor space, effective low-energy stirring, and reduction of measurement time. The calorimeter operates using the heat conduction principal with computer-controlled electrical compensation, which reduces the measurement time of each point from 10 to 3 min.