The proteinase, cathepsin L, has been strongly implicated in the processes of tumour invasion and metastasis. A new purification method, three-phase partitioning, characterised in terms of the parameters which affected its fractionation of proteins, was found to simplify the purification of cathepsin L from sheep liver. This method, together with a novel cation-exchange step on S-Sepharose and molecular exclusion chromatography, enabled the enzyme to
be purified to homogeneity, in a single-chain form. A further enzyme fraction was isolated as a proteolytically active complex with the endogenous inhibitor of cysteine proteinases, cystatin. Studies on the proteolytically active
complex revealed that approximately 60% of it was covalently bound and proteolytically active, while the other 40% was non-covalently bound and proteolytically inactive, in the manner normally found for the binding of cystatin to cysteine proteinases. A cystatin fraction from sheep liver containing variants of cystatin B, was shown to be able
to form complexes with free cathepsin L in vitro in a pH-dependent, rapid process, which was mildly stimulated by a reducing agent. Cathepsin L was also isolated from human spleen, but only as a protcolytically inactive complex, presumably also with cystatin(s). The complexed and free cathepsin L from sheep liver were analysed for their pH-dependent characteristics, and it was found that both forms of the enzyme were more active and stable at, or near, neutral pH, than would have been expected from published values. Specific polyclonal antibodies to pure sheep cathepsin L were raised in rabbits and chickens. The chicken egg yolk antibodies were of a much higher titre and were immunoinhibitory towards the enzyme, which the rabbit antibodies were not. Anti-peptide antibodies, raised in rabbits against a peptide sequence selected from the active site of human cathepsin L, were highly specific for cathepsin L and immunoinhibitory towards the enzyme. Together with the polyclonal anti-cathepsin L antibodies, they show promise for immunoinhibitory and immunocytochemical studies on the enzyme, and as potential anti-tumour
drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9536 |
| Date | January 1990 |
| Creators | Pike, Robert Neil. |
| Contributors | Dennison, Clive. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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