title: Purification and characterization of Plasmodium yoelii adenosine deaminase.
abstract: Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 μM and 34 μM for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, Post Box No. 173, Chatter Manzil Palace, Lucknow 226001, India.
authors.forename: Sarika
authors.initials: S
authors.lastname: Yadav
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
authors.forename: U N
authors.initials: UN
authors.lastname: Dwivedi
mesh_terms: D000243:Adenosine Deaminase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D058892:Adenosine Deaminase Inhibitors / Q000494:pharmacology; D000645:Ammonium Sulfate; D000818:Animals; D000918:Antibody Specificity; D000962:Antimalarials / Q000494:pharmacology; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010967:Plasmodium yoelii / Q000201:enzymology*; D011687:Purines / Q000494:pharmacology; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D058892:Adenosine Deaminase Inhibitors; D000962:Antimalarials; D011687:Purines; D000243:Adenosine Deaminase; D019791:Guanidine; D000645:Ammonium Sulfate