Effects of organophosphates on neural and purified liver tissue transglutaminase

Muñoz, D, 2010. Effects of organophosphates on neural and purified liver tissue transglutaminase. PhD, Nottingham Trent University.

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Abstract

Transglutaminase 2 (TGase 2) is a multifunctional calcium dependent enzyme that catalyzes protein modifications. TGase 2 is essential in neuronal cell differentiation and it has been reported that certain organophosphates are able to inhibit this process, and the organophosphate phenyl saligenin compound also disrupts TGase 2 activity. It has also been shown that the organophosphates chlorpyrifos (CPF) and chlorpyrifos oxon (CPFO), which cause developmental neurotoxicity, provoke several changes in differentiating rat C6 glioma cells at different levels. The aims of this thesis were to analyse the effects of CPF and CPFO on the TGases present in differentiating rat C6 glioma cells, to develop a new method for the purification of TGase 2 from guinea pig liver, to study possible direct interactions between TGase 2 and esterase inhibitors and to analyze a possible pathway for the externalisation of TGase 2.

In the presence of sodium butyrate, rat C6 glial cells differentiated into an astrocyte phenotype. Differentiation of the cells was associated with an increase in the activity, protein levels and gene expression of TGase 2. Differentiation in the presence of CPF or CPFO generated an increase in the activity of TGase 2, a decrease in its levels of gene expression but had no effect on the protein levels. These effects could be associated with a direct interaction between the organophosphates and TGase 2.

Chromatographic methods were developed to purify TGase 2 from guinea pig liver and the most effective one was a combination of ion exchange chromatography, protamine sulfate precipitation and hydrophobic interaction chromatography (HIC). The level of purity and yield obtained were superior to that of previously published methods. Furthermore, the final step of HIC could be applied directly to commercially available TGase 2 for the production of a highly purified TGase 2 sample.

When TGase 2 purified in this manner was assayed in the presence of CPF and CPFO, enzyme activity was observed to increase significantly, suggesting a direct interaction with TGase 2. By contrast, phenyl saligenin phosphate was found to inhibit TGase activity in vitro, which suggests a direct effect that may involve a different binding site and/or mechanism to CPF or CPFO. The aspartyl protease inhibitor pepstatin A was also able to inhibit directly TGase activity in vitro.

The final part of the project involved a short study of the potential association of TGase 2 with exosomes, in order to determine whether the latter might present a means of externalization of this enzyme. Exosomes purified from mouse N2a neuroblastoma cells were found to contain TGase 2, but its localization within the vesicles remains unclear.

Item Type:	Thesis
Creators:	Muñoz, D.
Date:	2010
ISBN:	9781369326536
Identifiers:	Number Type PQ10290404 Other
Divisions:	Schools > School of Science and Technology
Record created by:	EPrints Services
Date Added:	09 Oct 2015 09:35
Last Modified:	20 Jul 2021 15:25
URI:	https://irep.ntu.ac.uk/id/eprint/253

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