Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Abstract

Lee, D, and Moffatt, B. A. 1993. Purification and characterization of adenine pbosphoribosyltransferase from Arabidopsis ihaliana. -Physiol, Plant, 87: 483-492, Adenine phosphoribosyltransferase (APRT; EC 2.4.2,7) from Arabidopsis ihaliana was purified approximately 3800-foid. to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, {NHjjiSOj precipitation, Sephadex G-2S salt separation, ultracentrifugation and liquid chromatography on Diethyiaminoethyl Sephacel, Phenyi 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 3(X) nmol (mg total protein) ' min '. 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 (Mn-' or Mg-*). In the presence of MnCI,, other divalent cations (Mg-*, Ca-*, Ba-*, Hg-* and Cd-*) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K^ for adenine was 4.5±1.5 (lA/, the K^ for PRPP was 0.29±0.06 mM and the K, for PRPP was L96±0.45 mM. Assays using radiolabelled eytokinins showed thai purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K^ for benzyladenine was approximately 0.73+0.06 mM.

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…Assuming that exogenously fed CKs are metabolized in a mechanism similar to the endogenous hormone, these results suggest that APT1 can contribute to the interconversion of CK bases to CK nucleotides in vivo. Kinetic characterization of homogeneous APT1 activity in vitro also suggested that it is capable of utilizing CK substrates, although its affinity for BA is quite low, based on a K M of 730 mM for this substrate (Lee and Moffatt, 1993). As mentioned above, further characterization of the profile of CKs in this mutant will be necessary to elucidate whether it APT1 contributes to their interconversion.…”

Arabidopsis protein repair l‐isoaspartyl methyltransferases: predominant activities at lethal temperatures

“…We note that an optimal catalytic activity at lethal temperatures is also found for other enzymes in Arabidopsis . Ribulose‐1,5‐bisphosphate carboxylase/oxygenase and adenine phosphoribosyltransferase have optimal enzyme activity of at least 60°C (Salvucci et al 2001) and 65°C (Lee and Moffatt 1993), respectively. Additionally, it has been speculated that ribulose‐1,5‐bisphosphate caboxylase/oxygenase activase may be alternatively spliced as a result of environmental regulation because evidence shows a marked difference in temperature optima between its isoforms (Crafts‐Brandner et al 1997).…”

Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis

“…After centrifugation (10 min, 20,000g, 48C) the supernatant was used for the determination of enzyme activity. Measurement of salvage pathway enzymes was performed as given by Moffatt et al (2000) with [5-3 H]-uridine as substrate for UK and [2-14 C]-uracil as substrate for determination of UPRT activity according to the method of Lee and Moffatt (1993).…”