Biomedical subjects

P G Varley

Publications and source records attributed to P G Varley.

8 recordsLinked to original sources

PubMed1997

A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development.

Analytical ultracentrifugation (AUC) has reemerged as a powerful technique for protein characterisation. We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1 alpha (MIP-1 alpha) as a protein therapeutic. MIP-1 alpha has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers. Using AUC as a screening technique, we have produced a biologically active variant of MIP-1 alpha, BB-10010, which has a reduced tendency to aggregate in physiological buffers. The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC). Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association. This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies.

Biopharmaceutics↗

PubMed1995

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Amino Acid Sequence↗

PubMed1994

Fluorescence spectroscopy.

This chapter has introduced the theory of fluorescence, and briefly shown that it is a technique with many wide and varied applications in biochemistry. It has the overall advantage of being able to carry out measurements on small amounts of sample in solution, often revealing important information concerning the structure and dynamics of these systems.

Anisotropy↗

PubMed1993

Characterization of the solution properties and conformation of pneumolysin, the membrane-damaging toxin of Streptococcus pneumoniae.

Pneumolysin is a membrane-damaging toxin produced by Streptococcus pneumoniae. In order to understand fully the mode of action of this toxin, it is necessary to have an appreciation of the size, self-association behaviour and solution conformation of pneumolysin. A combination of analytical ultracentrifugation methodologies has shown that pneumolysin lacks self-association behaviour in solution and has provided a weight-average M(r) (M omega) of 52,000 +/- 2000, which was in agreement with that derived from the amino acid sequence. By determining a sedimentation coefficient (S20,w0) of 3.35 +/- 0.10 S, it was possible to suggest a model for the gross solution conformation of pneumolysin monomers. Spectroscopic methods provide additional secondary and tertiary structure information.

Amino Acids↗

PubMed1993

Characterization of the quaternary structure and conformational properties of the human stem cell inhibitor protein LD78 in solution.

The human LD78 protein (sometimes referred to as human macrophage inflammatory protein-1 alpha) has been shown to protect multipotential hemopoietic stem cells from the effects of cytotoxic agents. Administration of the recombinant stem cell inhibitor molecule LD78 as an adjunct to chemotherapy has potential clinical benefit in reducing or preventing the neutropenia associated with this treatment. At physiological ionic strength, the 8-kDa LD78 molecule exists as soluble, heterogeneous, multimeric complexes of mass ranging from 100 to > 250 kDa. The hydrodynamic and structural properties of LD78 have been determined in various buffer solutions using analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy. The results demonstrate that defined, homogeneous monomer and tetramer forms of LD78 can be prepared which display distinct conformational properties. The combined use of hydrodynamic and spectroscopic analysis provides an insight into the pathway and molecular mechanics of LD78 self-association.

Acetates↗

PubMed1992

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.

Acrylamide↗

PubMed1991

Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus.

3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity. It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein. As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature. Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature. These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins. They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity.

Acrylamide↗

PubMed1991

Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide.

The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophans, has been resolved into two approximately equal components, one accessible and one inaccessible to the relatively inefficient quencher succinimide. The inaccessible component is blue-shifted and exhibits a heterogeneous fluorescence decay which has a temperature-dependence and steady-state acrylamide quenching properties typical of a single tryptophan in a buried environment. This component is therefore assigned to the buried tryptophan W333. The presence of succinimide greatly simplifies the fluorescence allowing the conformational dynamics of the buried tryptophan and its environment to be studied without interference from the other tryptophan.

Calorimetry↗