Biomedical subjects

C Bardelle

Publications and source records attributed to C Bardelle.

3 recordsLinked to original sources

PubMed1999

Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1993

Membrane binding kinetics of factor VIII indicate a complex binding process.

Factor VIII functions as a component of the tenase enzyme complex upon phospholipid membranes. Factor VIII binds to phosphatidylserine-containing membranes and apparently provides high affinity binding sites for factor IXa upon these membranes. We have characterized the binding kinetics of human factor VIII with phosphatidylserine-containing membranes and directly compared the measured properties with those of factor V. The initial phase of association was evaluated in a stopped-flow apparatus by fluorescence energy transfer from aromatic residues in the protein to dansyl-labeled phosphatidylethanolamine in the vesicles. Association proceeded at an apparent second-order rate of 0.12 microM-1 s-1 for extruded phospholipid vesicles and 0.42 microM-1 s-1 for sonicated vesicles under pseudo-first-order conditions in which the phospholipid concentration determined the rate. Increased temperature resulted in more rapid association, and the effect decreased in the order extruded vesicles > sonicated vesicles > extruded vesicles of dioleoylphospholipids, indicating that the structure of the phospholipid membrane contributes to the activation energy of binding. The binding of fluorescein-labeled factor VIII to membranes supported on glass microspheres (lipospheres) was monitored by flow cytometry. Under conditions in which the factor VIII concentration determined the rate there was rapid initial association at 6.9 microM-1 s-1, accounting for half of the bound factor VIII, and a slower component of 0.87 microM-1 s-1, accounting for the other half. Likewise, the dissociation of factor VIII from liposphere membranes was biphasic with a faster component of 0.010 s-1 and a slower component of 0.0012 s-1. Rates of association and dissociation for factor V were similar to those for factor VIII and were biphasic. These results allow estimation of the size of the phospholipid sites that interact with factors VIII and V and suggest that both proteins bind to membranes via a multistep process in which rapid association is followed by a slower step yielding higher affinity binding.

Animals↗

PubMed1991

Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain.

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.

Amino Acid Sequence↗