Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800‐fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4)2SO4 precipitation, Sephadex G‐25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL‐4B, Blue Sepharose CL‐6B and adenosine 5′‐monophosphate‐Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)‐1 min‐1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+). In the presence of MnCl2+ other divalent cations (Mg2+, Ca2+, Ba2+, Hg2+ and Cd2+) inhibited the APRT reaction. Kinetic studies indicated that 5‐phosphoribose‐1‐pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μM, the Km for PRPP was 0.29±0.06 mM and the Ki for PRPP was 1.96±0.45 mM. Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 mM