Different location in dark‐adapted leaves of Phaseolus vulgaris of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and 2‐carboxyarabinitol 1‐phosphate

Abstract

In situ RuBisCO activity was analyzed in order to determine whether 2-carboxyarabinitol 1-phosphate (CAIP) interacts with the enzyme in dark-adapted leaves of Phaseolus vulgaris. Leaves ground to fine powder in liquid nitrogen were put directly into a reaction mixture containing a saturating concentration of ribulose 1,5-bisphosphate (RuBP) to preserve the activity of RuBisCO which was in the chloroplasts. Some 70% of the total catalytic sites of RuBisCO possessed carboxylase activity in this assay, however, RuBisCO was inhibited in the absence of RuBP. CA1P seemed to be concentrated in the veins. These results indicate that RuBisCO was not complexed with CAIP in leaves.

The localisation of 2‐carboxy‐d‐arabinitol 1‐phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L

“…This was interpreted as meaning that the inhibitor was mainly in the veins. Reported below are experiments repeating the approach described above [7] with a commercial variety of Phaseolus vulgaris recently used in investigations of the biosynthesis of CA1P [8]. The results con£ict with those of Anwaruzzaman et al [7].…”

“…Reported below are experiments repeating the approach described above [7] with a commercial variety of Phaseolus vulgaris recently used in investigations of the biosynthesis of CA1P [8]. The results con£ict with those of Anwaruzzaman et al [7]. An explanation for the con£icting results and conclusions was sought and may be entailed in the exact conditions used to produce dark-adapted plants and the extent to which they were exposed to light during collection of samples.…”

“…The subsequent demonstration that both CA1P and a speci¢c phosphatase that degrades it were located in the chloroplast [5,6], and that CA1P could be released from Rubisco by Rubisco activase, gave further support to this hypothesis. In contrast, more recent claims by Anwaruzzaman et al [7] that CA1P and Rubisco are not in the same part of the leaf and that CA1P only becomes bound during extraction is inconsistent with a role for CA1P in Rubisco regulation and needs further examination.…”

“…The crucial experiment, upon which the claim [7] depended, involved the rapid freezing of dark-adapted leaves in liquid N P , grinding the tissue to a powder, and measuring the Rubisco activity released as the frozen powder thawed and dispersed in a reaction mixture containing appropriate substrates and cofactors. This was referred to as the`direct assay'.…”

“…This was consistent with the conclusion that the inhibitor and enzyme were initially separated in the frozen tissue but became associated with each other during the time needed to make conventional extracts. Vein-enriched tissue [7] was prepared from dark-adapted leaves by digesting with cellulase and pectinase to release mesophyll cell protoplasts. The ¢brous residue remaining, referred to as vein-enriched tissue, was extracted with acid and the inhibitory e¡ect of components of the extract on Rubisco activity compared to components in a similar extract of intact leaves.…”