Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598993
Title: Signalling in Trypanosoma b. brucei mediated by GTP-binding proteins and phosphorylation
Author: Fernandez, D. S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Abstract:
Antisera raised against the C-terminal decapeptide of Gsα, (RMHLRQYELL or RM), immunodetected polypeptides of 52, 45 and 42 kDa species consistent with identified mammalian species. The specificity of the immunoreactivity was demonstrated by competition with preincubations utilising the decapeptide RM against which the antiserum was raised. Antibodies to Gi and Gq and their respective competing peptides were also utilised. These indicated the presence of a 41 kDa polypeptide cross-reactive with anti-Gi and two polypeptides of 43 and 42 kDa cross-reactive with anti-Gq in trypanosome extracts. GTP binding proteins of T. b. brucei were additionally identified by [α32P]-GTP binding. This method elucidated polypeptides of 65, 52, 45 and 41 kDa as well as low molecular weight polypeptides (less than 30 kDa) typical in size and abundance of members of the ras superfamily. GTP-binding proteins were also labelled by a photoaffinity reaction by UV irradiation in the presence of [α32P]-GTP, identifying polypeptides of 160, 140, 65, 52 and 45 kDa in trypanosomes which co-localised with CS1-immunoreactive species. Cholera toxin catalysed ADP-ribosylations of 52 and 45 kDa polypeptides while pertussis toxin mediated the ADP-ribosylation of 41-43 kDa polypeptides; consistent with observations for mammalian species and the immoreactivity data presented. The role of tyrosine kinase signalling pathways was also studied. By stimulating conditions required for transformation of the bloodstream form to the procyclic, increases in autophosphorylating kinase activity and tyrosine phosphorylation were observed. Phosphorylations on polypeptides of 138, 87, 66, 60 and 46 kDa were found to be alkaline stable, identifying them as possible tyrosine kinases. Phosphoamino acid analysis confirmed phosphorylation of tyrosine of polypeptides of 138, 66 and 46 kDa. The autophosphorylating kinase activity of a 138 kDa polypeptide was found to be 1) stimulated by EGF; 2) shown to be alkaline resistance and; 3) to be phosphorylated on tyrosine which indicate it might be the putative EGF receptor tyrosine kinase of T. brucei while a 98 kDa polypeptide was identified by immunoblotting as a possible insulin receptor kinase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.598993  DOI: Not available
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