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Isolation of ribulose bisphosphate carboxylase‐oxygenase from non‐hardened and hardened needles of Pinus sylvestris
Author(s) -
Gezelius Kerstin,
Widell Anna
Publication year - 1986
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.1399-3054.1986.tb02443.x
Subject(s) - pyruvate carboxylase , oxygenase , ribulose 1,5 bisphosphate , chemistry , chromatography , isoelectric focusing , biochemistry , rubisco , sucrose , enzyme , botany , biology
Ribulose bisphosphate carboxylase‐oxygenase, RuBP carboxylase (EC 4.1.1.39), was purified from non‐hardened and hardened needles of Pinus sylvestris L. Needles were collected from pine seedlings cultivated in nutrient solution in a climate chamber from seedlings grown outdoors, and from a tree in a natural stand. The enzyme was isolated from crude extracts through quantitative precipitation in polyethylene glycol 4000 and MgCl 2 , followed by sucrose gradient centrifugation in a fixed angle rotor. The purified enzyme seemed homogeneous by the criterion of (sodium dodecylsulphate) polyacrylamide gel electrophoresis. Contamination by nucleic acids was negligible. The RuBP carboxylase protein content of the gradient fractions was estimated as A 280 1 cm × 0.61 mg ml −1 . Carboxylase activities were determined in a radioactive assay at 25°C. The specific activity of RuBP carboxylase isolated from non‐hardened needles was approximately 1 μmol CO 2 (mg protein) −1 min −1 . For enzyme isolated from hardened needles collected during winter the specific activity was somewhat lower due to loss of enzyme activity during the preparation. The described two‐step procedure provides a means for quantitation of the RuBP carboxylase protein in pine needles during all seasons.