Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani.
The adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activities from promastigotes of Leishmania donovani have been purified to homogeneity using ammonium sulfate precipitation, DEAE-cellulose exclusion, and either AMP-agarose (APRTase) or GTP-agarose (HGPRTase) affinity chromatography. The specific activities of the affinity-purified APRTase and HGPRTase fractions were 326-fold and 1341-fold greater than those in the 40-80% ammonium sulfate precipitate, respectively. The purified APRTase migrated as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels with a size of 29 kDa, while HGPRTase was also determined to be homogeneous by SDS gel electrophoresis with a size of 24 kDa. In addition, a mutant cell line, APPB2, partially deficient in APRTase activity, still contained quantities of purifiable APRTase protein, while a clonal secondary derivative of the APPB2 cell line that is completely deficient in APRTase activity, APPB2-640A3, failed to express purifiable APRTase protein. The homogeneous enzymes possessed apparent Km values for their nucleobase substrates between 2.0 and 5.0 microM, and both enzymes were inhibited by their immediate or ultimate reaction endproducts, APRTase by AMP and PPi and HGPRTase by GMP, GTP, and PPi. The generation of homogeneous preparations of APRTase and HGPRTase protein will serve as a prerequisite for the generation of immunological and molecular biological probes to analyze the leishmanial phosphoribosyltransferases.
Allen
T
T
Department of Biochemistry, Oregon Health Sciences University, Portland 97201.
Henschel
E V
EV
Coons
T
T
Cross
L
L
Conley
J
J
Ullman
B
B
D000228:Adenine Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007041:Hypoxanthine Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D007893:Leishmania donovani / Q000201:enzymology*; D010430:Pentosyltransferases / Q000302:isolation & purification*; D013379:Substrate Specificity
D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase