Dennison, Clive.Dehrmann, Frieda Marie.2013-10-212013-10-2119981998http://hdl.handle.net/10413/9777Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a novel, scaled-up protein purification method and purified to homogeneity in the single-chain form. In addition, the high molecular weight variant of cathepsin L covalently complexed with stefin B was isolated. Both cathepsin L and the complex were stable, in respect of their proteolytic activity, to the chaotropic agent urea, both showing enhanced activity in the presence of urea. Urea did not dissociate the complex. The suitability of cathepsin L for a purported extracellular role was addressed by investigating its pH optimum and pH stability. Cathepsins L and B are affected by ionic strength and so buffers of constant ionic strength (rather than constant molarity, and therefore varying ionic strength) were used in determining their pH optima and stability. Cathepsins L and B had apparent pH optima of pH 6.5 and 7.5, respectively, (measured with synthetic substrates) and, contrary to the previous belief, were substantially stable at physiological pH. In Hanks' balanced salt solution, a model of the extracellular fluid, they were shown to be active and stable, cathepsin L having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumour pH). It was also shown that prior reductive activation of these enzymes increased their stability to extracellular conditions, supporting the hypothesis that the active site thiolate-imidazolium ion pair contributes to their stability. The nature of the bond between cathepsin L and stefin B in the covalent complex was examined, using CNBr cleavage, HPLC and amino acid sequencing. Stefin B was shown to be associated with residues 1-137 of cathepsin L via a reduction sensitive linkage which was deduced to be a thioester bond betwen Asp-71 of cathepsin L and Cys-3 of stefin B. Polyclonal antibodies to cathepsin L and stefin B-complexed cathepsin L were raised in rabbits and chickens, and characterised with respect to their suitability for immunocytochemical localisation of these forms of cathepsin L.en-ZACathepsin L.Proteolytic enzymes.Cancer invasiveness.Theses--Biochemistry.Thesis