Immunochemical and catalytic characteristics of adenosine kinase from Leishmania donovani.
Polyclonal antibodies to homogeneous preparation of adenosine kinase from Leishmania donovani were raised in rabbit. The antiserum was inhibitory and precipitated enzyme activity from both homogeneous and partially purified adenosine kinase from the parasite. However, the antiserum did not immunoprecipitate adenosine kinase of other higher eukaryotic sources tested so far. Immunoblot analysis of extracts from L. donovani and other sources revealed specific reaction of the antiserum with only the parasite enzyme. Under similar conditions, the enzyme monophosphorylated adenosine and 7-amino-3[beta-D-ribofuranosyl]-1H-pyrazolo[4,3-d]pyrimidine (formycin A) with almost equal efficiency, exhibiting Km values of 16 and 24 microM, respectively. The turnover number (Kcat) of the enzyme with both adenosine and formycin A was 24 s-1, whereas Kcat/Km yielded values of 1.5 and 1.0 microM-1 s-1, respectively. Substrate competition experiments indicated strong inhibition of [3H]formycin A phosphorylation by adenosine. In contrast, [3H]adenosine phosphorylation was insensitive to formycin A except at very high concentrations. The inhibitions of [3H]formycin A and [3H]adenosine phosphorylation by adenosine and formycin A were noncompetitive with respect to each other. Of the two nucleosides, adenosine was found to be effective in eluting the enzyme from the 5'-AMP Sepharose 4B column. Phosphorylation of [3H]formycin A was strongly inhibited by N-ethylmaleimide at concentrations which exerted minimal effect on [3H]adenosine phosphorylation. Adenosine exclusively, but not formycin A, protected the enzyme from N-ethylmaleimide-mediated inactivation. Taken together the results suggest that (a) adenosine kinase from L. donovani is immunologically distinct and (b) the enzyme possibly has two discrete catalytically active nucleoside interacting sites.
Bhaumik
D
D
Leishmania Group, Indian Institute of Chemical Biology, Calcutta.
Datta
A K
AK
D000241:Adenosine / Q000378:metabolism / Q000494:pharmacology; D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D005033:Ethylmaleimide / Q000494:pharmacology; D005573:Formycins / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D013045:Species Specificity
D005573:Formycins; D011189:Potassium Chloride; C034550:formycin; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000248:Adenosine Kinase; D000241:Adenosine; D005033:Ethylmaleimide