| dc.description.abstract | The genetic diversity in 13 stocks and clones of T. vivax from East and West Africa was compared by isoenzyme analysis of 13 enzymes. The Ugandan and West African stocks and clones showed a very high degree of genetic similarity to each other but they differed from the Kenyan stocks and clones. Thus, our sample could be divided into two: a Kenyan group, and a Ugandan and West African
group.
The phenotypic diversity of these stocks was further investigated with respect to their banding patterns for peptidases, an isoenzyme, on sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS -PAGE). All the stocks and clones examined revealed a common band at about 92 kDa and an additional band at about 200 KDa or 220 kDa. Kenyan stocks and clones revealed bands at 92kDa and 200 kDa while Ugandan and West African stocks and clones revealed bands at 92 kDa and 220 kDa. The observations were consistent with the results obtained from the isoenzyme analysis. An aminopeptidase was purified from a clone of T. Vivax (ILDat 2.1) with a purification factor of 226. Peptidase activity was observed at two molecular weights- about 92 kDa and 200 kDa on SDS-polyacrylamide gels stained for peptidase activity. The purified fraction revealed additional bands at about 46 kDa, 69 kDa and about 150 kDa on silver-stained SDS-polyacrylamide gels. The enzyme hydrolysed N-leucyl-amides and N-leucyl-peptidts. An N-phenylalanyi-peptide was
also hydrolysed but much more slowly than the equivalent leucyl compound. The enzyme is optically specific, preferring peptides with the first two amino acids in the L-configuration. It would not cleave substrates with blocked N-terminal amino groups such as N-benzoyl-L-leucyl-L-tyrosinamide. The aminopeptidase has an isoelectric point of pH 7.5 and a pH optimum of 8.5. Using the substrate L-teucyl-L-glycylglycine, the enzyme had a Vmax of 4.1 (± 0.4) 10⁻⁴ mmol min⁻¹ and a Km of 34 ± 7 mM. Amastatin and bestatin, known aminopeptidase inhibitors, inhibited peptidase activity. Bestatin at concentrations of 33 µM and 250 µM gave inhibitions of 23% and 46% respectively. Amastatin at concentrations of 33 #1114 and pM gave inhibitions of 23% and 46% respectively. Parasite peptidase could not be detected in the plasma of heifers infected with a haemorrhagic stock of T. vivax by sandwich assay (ELISA) or on starch gels stained for peptidase activity. Rabbit antiserum produced to ILD at 2.1 aminopeptidase localized the enzyme in sections of homologous and heterologous parasites to the Iuminal portion of large lysosome-like vesicles lying between the nucleus and the flagellar pocket, often in close proximity to the nucleus. From these findings we speculate that parasite leucine aminopeptidase could be involved in the cleavage of the C-terminal hydrophobic tail of trypanosome VSGs which terminates as Leu-Leu-Leu or Leu-Leu-Phe. The peptidase could therefore be a potential target for drug development. | en_US |
