Organophosphate hydrolases as catalytic bioscavengers of organophosphorus nerve agents.

Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection.

Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning.

Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration.

Eukaryotic DING proteins are endogenous: an immunohistological study in mouse tissues.

Background: DING proteins encompass an intriguing protein family first characterized by their conserved N-terminal sequences. Some of these proteins seem to have key roles in various human diseases, e.g.

Integrative analytical approach by capillary electrophoresis and kinetics under high pressure optimized for deciphering intrinsic and extrinsic cofactors that modulate activity and stability of human paraoxonase (PON1).

Paraoxonase (PON1) is working in vivo in a particular dynamic environment including HDL particles and associated molecules. To decipher the respective and/or concomitant role of the different cofactors involved in this molecular organization, an approach using multiple experimental techniques based on capillary electrophoresis and classical kinetics or kinetics under high pressure was implemented. The effects of calcium and phosphate as protein or plasma cofactor, of human phosphate binding protein (HPBP) as enzyme chaperone, and of a PON1 inhibitor as an active site stabilizer, on the catalytic activities and functional oligomerization of PON1 were scrutinized.

Exploring the structural and functional stabilities of different paraoxonase-1 formulations through electrophoretic mobilities and enzyme activity parameters under hydrostatic pressure.

Human paraoxonase-1 (HuPON1) is the ideal candidate to engineer as catalytic bioscavenger for pre-treatment and therapy of exposure to toxic organophosphorus compounds. HuPON1 is a naturally-occurring hydrophobic plasma protein associated with a partner, the human phosphate binding protein (HPBP) on high density lipoproteins. The relationships between the composition and the size of multimeric states of HuPON1 are not well understood.

Tandem use of X-ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38-kDa apolipoprotein.

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal residues, are systematically absent from eukaryotic genome databases.

Stability of highly purified human paraoxonase (PON1): association with human phosphate binding protein (HPBP) is essential for preserving its active conformation(s).

The biological role of human paraoxonase (PON1) remains unclear, whilst there is a consensus that the enzyme has a protective influence. A toxicological role, protecting from environmental poisoning by organophosphate derivatives drove earlier works, and more recently, clinical interest has focused on a protective role in vascular disease. PON1 resides essentially on HDL particles, a complex and dynamic molecular environment.

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography.

Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations.

Serendipitous discovery and X-ray structure of a human phosphate binding apolipoprotein.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes.

Crystallization and preliminary X-ray diffraction analysis of human phosphate-binding protein.

Human phosphate-binding protein (HPBP) was serendipitously discovered by crystallization and X-ray crystallography. HPBP belongs to a eukaryotic protein family named DING that is systematically absent from the genomic database. This apoprotein of 38 kDa copurifies with the HDL-associated apoprotein paraoxonase (PON1) and binds inorganic phosphate.

Capillary electrophoresis versus differential scanning calorimetry for the analysis of free enzyme versus enzyme-ligand complexes: in the search of the ligand-free status of cholinesterases.

Cholinesterases (ChEs) are highly efficient biocatalysts whose active site is buried in a deep, narrow gorge. The talent of CE to discover inhibitors in the gorge of highly purified preparations has fairly altered the meaning of a ChE ligand-free status. To attempt at a description of this one, we investigated the stability of Bungarus fasciatus acetylcholinesterase (AChE), alone or complexed with different inhibitors.

Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence.

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties.

Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1).

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents.

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation.

Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters.

The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability.

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366-14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co(2+)/Co(2+)-PTE.