Connolly, Christopher Neil Peter; (1993) Development of an enzymatic probe for the exocytic pathways of animal cells. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The polarised epithelial cell phenotype is generated and maintained by the polarised delivery of proteins to either the apical or basolateral domains, where they perform domain-specific functions. Proteins destined for the two domains have been co-localised up to and including the trans-Golgi network. Subsequent transport to either domain occurs by distinct vesicles, although the morphology and itinerary of these pathways are not understood. This lack of understanding is due mainly to the low sensitivity of antibody detection, resulting from loss of antigenicity and inaccessibility. Thus, advancements in this area await the development of a more sensitive detection system. A solution to this problem would be the development of an enzymatic probe within the exocytic pathway of animal cells. The aim of this research project was to develop such a probe. Three potential probes were examined, microperoxidase, β-galactosidase and horseradish peroxidase (HRP). Unfortunately, microperoxidase expressed from DNA was inactive. However, the other two probes were both found to be active and present within the exocytic pathway. Horseradish peroxidase activity was further analysed and found to be secreted from cells with a t1/2 of 80 minutes. The development of stable cell lines expressing HRP have proved difficult, with cells dying after a only few passages. This cell death was concomitant with the intracellular accumulation of inactive HRP. Interestingly, the addition of exogenous haemin converted this inactive intracellular pool to an active and secreted form of HRP. It may now be possible to rescue stable cell lines by their incubation in the presence of low levels of haemin. In summary, an enzymatic probe has been successfully introduced into the exocytic pathway of animal cells. This probe (HRP) can be detected at high levels within cells by electron microscopy. Enzyme activity may also be detected biochemically by the spectroscopy of soluble reaction product.

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