A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco.

Abstract:

:Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.

journal_name

Photosynth Res

journal_title

Photosynthesis research

authors

Scales JC,Parry MA,Salvucci ME

doi

10.1007/s11120-013-9964-5

subject

Has Abstract

pub_date

2014-03-01 00:00:00

pages

355-65

issue

3

eissn

0166-8595

issn

1573-5079

journal_volume

119

pub_type

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