The main site for protein degradation along the endosomal pathway is believed to be the late
endosome. Lysosomes are thought to be storage organelles that, when necessary, inject
proteases into the late endosome. It was hypothesised that differences in the lumenal redox
environments between the two organelles could be responsible for their functional
differences. In an attempt to quantify this potential difference, the lysosomal cysteine
protease cathepsin B was isolated by an improved purification procedure. Several
intracellular reducing agents were used to activate cathepsin B, the most effective being
cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to
model endosomal conditions. An inverse relationship was found between the pH and the
concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B
may have an optimal redox potential. In order to determine this potential, cysteinexystine
redox buffers were made up and used in determination of the activity of the enzyme against a
synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be
determined using either substrate, but it was found that cystine stimulated proteolysis of
haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain
hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote
substrate structure, effectively increasing the substrate concentration. These findings and
other results obtained from the literature have been used to create a model of how proteolysis
may be regulated along the endosomal system. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9781 |
| Date | 21 October 2013 |
| Creators | Pillay, Ché Sobashkar. |
| Contributors | Dennison, Clive. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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