Identification and Functional Aspects of Protein Kinase Activity in Leishmania Aethiopica

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Addis Ababa University


Protein kinase activity was demonstrated on intact Leishmania aethiopica promastigotes but not on heat generated "amastigotes". Similar work conducted on lysed cells revealed enzyme activity in soluble and particulate fraction of flagellated parasites. Enzyme on intact cell plasma membrane preferred Histone V-S over protamine as in vitro substrate whereas enzyme from soluble or particulate fraction of lysed cells displayed the reverse in vitro preference. On the other hand enzymes from both intact and lysed cells were inhibited by Staurosporine and Formycin ATP. Incubation of intact stationary phase parasites with t 2pJ ATP led to phosphorylation of eight protein bands. Similar experiment conducted using lysed cells revealed twenty bands. Incubation of the cells with [32pJ with the assumption of generating intracellular t2 P ] ATP also led to a patte111 of phosphorylation similar to that of lysed cells. It follows from the above that Leishmania aethiopica promastigotes possess both extra and intracellular endogenous substrates of protein kinases. Measurement of soluble kinase activity revealed increased activity as cells went from log to stationary phase where intact cell activity remained constant. Exposure to elevated temperature (37') induced transformation of the promastigotes to aff/agellated organism "amastigotes". This transformation resulted in a potent down regulation of both the soluble 'and the ecto kinase activities. In the promastigotes, Staurosporine caused morphological alterations. On the other hand Formycin ATP had a profound inhibitDlY effect on thymidine incorporation and cellular proliferation. These effects of Formycin ATP do not seem to be wholly accounted by its protein kinase inhibitory activity. A classical protein kinase C like activity could not be detected in the promastigotes. However, evidence for the probable presence of a phorbol ester insensitive, calcium and phospholipid independent protein kinase C is presented. All the same these observations do not rule out the presence of more than one subclass of protein kinases. This work suggests that major protein kinase activities may be involved in the regulation of proliferation, development and adaptation to "hostile" environment of Leishmania aethiopica promastigotes. It also argues that the continued study of key events in the biology of this organism is cTilcial in the development of better therapeuticstrategies.