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Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana
Author(s) -
Lee Diana,
Moffatt Barbara A.
Publication year - 1993
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1993.tb02497.x
Subject(s) - adenine phosphoribosyltransferase , chemistry , divalent , sephadex , chromatography , enzyme , biochemistry , pyrophosphate , sepharose , stereochemistry , purine , organic chemistry
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, (NH 4 ) 2 SO 4 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 (Mn 2+ or Mg 2+ ). In the presence of MnCl 2+ other divalent cations (Mg 2+ , Ca 2+ , Ba 2+ , Hg 2+ and Cd 2+ ) inhibited the APRT reaction. Kinetic studies indicated that 5‐phosphoribose‐1‐pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K m for adenine was 4.5±1.5 μ M , the K m for PRPP was 0.29±0.06 m M and the K i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K m for benzyladenine was approximately 0.73±0.06 m M