Isolation of ribulose bisphosphate carboxylase‐oxygenase from non‐hardened and hardened needles of Pinus sylvestris

Abstract

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 MgCl2, 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 A2801 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 CO2 (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.

Suboptimal nitrogen status sensitizes the photosynthetic apparatus in willow leaves to long term but not short term water stress

“…The slurry was filtered and centrifuged (40000g, 20min). The enzyme in the supernatant was purified, by sucrose gradient centrifugation, and quantified as described in (Gezelius and Widell 1986) except for the medium used: 0.05 M Tris-HC1-N a O H (pH 8 at 4 °C), 0.05 M NaHCO3,0.01 M MgC12, 1 mM EDTA, 1 mM fl-mercaptoethanol. The working range of the plates was defined with three lots of the standard (precipitate heights about 2.5, 3.2 and 4.2 cm) that in all cases could be fitted to a line with r 2 > 0.999.…”