Cathepsin D is a lysosomal acid proteinase that exists as different isoforms. This enzyme is found in most cell types, and macrophages have particularly high levels of this enzyme. Cathepsin D has been implicated in some disease related inflammatory processes that have also been associated with increased macrophage infiltration. The existance of isoforms of cathepsin D suggested the possibility that individual forms of this enzyme may have immunologically unique characteristics. Monoclonal antibodies to cathepsin D could aid in the study of the immunological characteristics of individual isoforms of this enzyme as well as provide a means by which to screen a variety of sample types for cathepsin D levels. In this study we generated a panel of monoclonal anti-cathepsin D antibodies and used these to screen isoelectrically separated cathepsin D samples for unique immunoreactivity patterns. We also used these antibodies to measure changes in the levels of intra- and extracellular cathepsin D that accompany monocyte to macrophage differentiation, and changes that occur in response to the treatment of melanoma cells with tumor necrosis factor (TNF) and interferon. We found that there are unique immunological characteristics associated with individual isoforms of human liver cathepsin D as well as cathepsin D from different cell types. We also observed increases in levels of this enzyme as monocytic cells differentiate to macrophage-like cells. This indicates that cathepsin may be involved in some of the proteolytic processes mediated by macrophages. Different inducers of differentiation resulted in different cathepsin 1D immunoreactivity patterns, suggesting that there may be isoform specificity dependent on the inducer. Cathepsin D levels also increase in neoplastic cells treated with TNF and/or interferon indicating that cathepsin D may play some role in the mechanism of action of the tumor inhibitory effects of these agents. The use of monoclonal antibodies provides assays for cathepsin D which can be used with a variety of sample types and further provides enhanced specificity and sensitivity relative to other assay systems used in the past.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Master of science Johannesburg. 1996 / An attempt has been made to purify and isolate a starch endo-hydrolase enzyme produced by Streptococcus sanguis, an organism that may be implicated in dental caries. In order to isolate the enzyme by affinity binding,& chemically modified amylopectin Was prepared, similar to the preparation of chromogenic substrate for the assay of a-arnylase, but without dye. The amylopectin was treated with 10 percent ammonium sulphate at 55°C for 45 minutes. A crude enzyme extract was prepared from concentrated culture medium and by precipitation with 60 percent ammonium sulphate. The concentrated culture medium was added to the modified amylopectin substrate and the mixture was incubate for 30 minutes at 37'C and centrifuged. The precipitate was resuspended in 20 mM bepes buffer pH 6.5 which contained 0.02 M KCI to release the enzyme from the enzyme-substrate complex. The suspension was tested for enzyme activity and the presence of proteins, More than 50 percent of the yield of the enzyme was achieved by this process, after three assays, from both crude enzyme extracts and enzyme serum samples. A 5.2 fold purification was obtained from the extraction process of the crude enzyme extract and a protective enzyme activity effect was noticed in the presence of ammonium sulphate. The analytical methods selected for the activity assay were mainly used for the activity evaluation of a-amylases and carbohydrate hydrolysing enzymes. The result showed carbohydrate interference. The isolation method proved sensitive and highly specific for the isolation of a starch metabolizing enzyme produced from Streptococcus sanguis 1MC 204. The purification of the enzyme by gel filtration and its characterization by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that this enzyme was a glycoprotein . SDS-PAGE was performed with mucin and heparin in the. presence of other. proteins as markers, and stained with the periodic acid- aldan prestained silver method. This gave one transparent-band around the phosphorylase b marker and four other more slowly running clear bands. Further, the comparison of scans of several proteins and glycoproteins with the scan of the eluted sample of the amylopectin extracted enzyme showed a similarity with the UV scan of mucin and confirmed that the enzyme was a glycoprotein. It may be further characterized by selecting methods that take into account the ambohydrate content of the protein and by eliminating the carbohydrate interference in the assays. 3

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Typescript. / Digitized by Kansas Correctional Industries

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by Nancy Wang. / Bibliography: leaves 126-144 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

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