Comparative studies of the purified cysteine proteases of trypanosoma brucei and trypanosoma congolense
| dc.contributor.author | Konde, Victor | |
| dc.date.accessioned | 2011-04-04T16:04:55Z | |
| dc.date.available | 2011-04-04T16:04:55Z | |
| dc.date.issued | 2011-04-04 | |
| dc.description.abstract | The cysteine protease enzymes from T. congolense and T. brucei were purified from pure blood stream forms of the trypanosomes grown in adult albino rats. The trypanosomes were harvested when the number of trypanosomes had reached 108/ml of infected blood or higher. The rats were supplied with 10% glucose in drinking water when the parasiteamia o was 10 /ml which enabled the rats to survive for at least two extra days. In case of T. brucei infections, the level of stumpy forms in blood was monitored and when the number of stumpy forms reached 50% or more the trypanosomes were harvested. This resulted in an increase in cysteine protease recovery by at least two fold. The enzymes were successfully purified using egg white cystatin immobilised on sepharose. There were no significant differences in the yields of the two cysteine proteases and both enzymes had similar activity. The activity of the two enzymes was assayed with carbobenzoxyphenylalaninyl- argininyl 7-amido-4-methylcoumarin by monitoring the fluorescence at 365 nm. The reactions were performed at pH 6 at room temperature (28 - 30°C). T. congolense cysteine protease was purified using a monoclonal antibody, prepared against the T. congolense cysteine protease, immobilised on sepharose. The enzyme was of similar molecular weight as that purified by the cystatin-sepharose column. The enzyme was incubated with a variety of host macromolecules, mainly connective tissue proteins and the digestion products were followed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). hi other cases the proteins were copolymerized in SDS-PAGE and then digestion monitored in zymograms. The enzymes expressed proteolytic activity against collagen type I, collagen type IV, glycoproteins, fibronectin, laminin and to a small extent against elastin. Fibronectin was the best substrate of all the connective tissue proteins while elastin was least reactive. All the above connective tissue proteins were not denatured prior to digestion as reported in similar experiments. The two trypanosome cysteine proteases failed to round off and detach cells in culture at our working enzyme concentration (160 ug/ml). The enzymes had greater effect on the cell membrane than on the intercellular matrix, an effect that is similar to papain. The enzymes have high proteolytic activity against cell surface proteins and this may account for the failure to round off cells, as the enzymes are likely to preferentially attack cell surface proteins rather than proteoglycans in the extracellular matrix. The enzymes were inhibited by trans-epoxysuccinyl-1-leucylamido (4-guanidino) butane (E-64), egg white cystatin, and Noc-p-tosyl-1-l-lysine chloromethyl ketone (TLCK) while phenylmethylsulphonyl-fluoride (PMSF) and aprotinin failed to inhibit the enzymes to any noticeable extent. The enzymes digested bovine serum albumin (BSA), fibrinogen and casein and their proteolytic products were resolved on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were resolved at similar regions and the patterns of bands produced by the two trypanosome cysteine proteases were identical. This strongly suggests that the trypanosome cysteine proteases under study have preference for the same peptide bonds. The two trypanosome enzymes have similar proteolytic properties and are cysteine proteases in nature as shown by their inhibition by cysteine protease inhibitors | en_US |
| dc.identifier.uri | http://dspace.unza.zm/handle/123456789/278 | |
| dc.language.iso | en | en_US |
| dc.subject | : Cysteine proteinases | en_US |
| dc.subject | : Trypanosoma brucei | en_US |
| dc.title | Comparative studies of the purified cysteine proteases of trypanosoma brucei and trypanosoma congolense | en_US |
| dc.type | Thesis | en_US |