Dear Fahimeh,

The attached publication covers the answer to your question.

1- Photosynth Res. 2014; 119(3): 355–365.

Published online 2014 Jan 5. DOI:  10.1007/s11120-013-9964-5

PMCID: PMC3923112

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

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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 remodeling 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.

Electronic supplementary material

The online version of this article (DOI:10.1007/s11120-013-9964-5) contains supplementary material, which is available to authorized users.

Keywords: Carbon metabolism, Enzyme regulation, Molecular chaperone, AAA+

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Introduction

Improving the catalytic or regulatory properties of Rubisco to increase the rate of carbon assimilation in photosynthesis has been suggested as a strategy for boosting crop yields (Parry et al. 2013). Increasing the turnover rate of Rubisco or its affinity and/or specificity for CO2 (Spreitzer and Salvucci 2002; Whitney et al.2011), preventing inactivation of Rubisco during periods of high temperature (Kurek et al. 2007; Parry et al.2011; Carmo-Silva and Salvucci 2012) or improving the response time of Rubisco activation during transitions in light intensity (Carmo-Silva and Salvucci 2013) have all been proposed as possible ways to enhance the carbon fixing step of photosynthesis. To determine the effects of naturally-occurring or artificially-introduced modifications of Rubisco on carboxylation activity or the interaction with the catalytic chaperone, Rubisco activase (RCA), it is important to have a reliable method for measuring Rubisco and RCA activity. Ideally, the assay should be amenable to high throughput measurement of activity in plant tissue and with purified proteins. Given the central role of RCA in controlling the activation state of Rubisco, it is also desirable that the assay can measure RCA activity in response to variable ratios of ADP: ATP. The ratio of these adenine nucleotides is the major physiological factor affecting RCA activity (Robinson and Portis 1989a; Carmo-Silva and Salvucci 2013).

file:///C:/Users/PC/Downloads/JournalofBiochemicalandBiophysicalMethodsVolume52issue32002doi10.1016_s0165-022x0200072-6SubhraChakrabartiSumanaBhattacharyaSanjoyKBhattacharya--Anonradioactiveassay.pdf

file:///C:/Users/PC/Downloads/PlantCellEnvironmentVolume30issue92007doi10.1111_j.1365-3040.2007.01679.xRONANSULPICEHENDRIKTSCHOEPMARIAVONKORFFDIRKBSSISB--Descriptionandapplicationsofarapidand.pdf

You could follow the production of glycerate 3-phosphate from ribulose bisphosphate by liquid chromatography/mass spectrometry or NMR. If that equipment is unavailable, you could probably do it less quantitatively using thin layer chromatography.

If you can get the enzyme 3-phosphoglycerate kinase, you could use a coupled enzyme system, whereby the glycerate 3-phosphate +ATP is converted by 3-phosphoglycerate kinase to 1,3-bisphosphoglycerate + ADP, then measure the production of ADP from ATP using any of various systems. This might be pretty complicated to set up, however.

If you are measuring the O2 consumption reaction to produce 2-phosphoglycolate and 3-phosphoglycerate, you could just use a Clark electrode and measure the depletion of oxygen. If you are measuring the CO2 consumption reaction, you could do a coupled reaction by adding excess 3-phosphoglycerate dehydrogenase and NAD+, which you monitor spectrophotometrically at 340nm. I don't know the optimal pH or any metals requirements for that enzyme off the top of my head, but I wouldn't be surprised if phosphate buffer was a poor choice, due to the probability of the phosphate being a competitive inhibitor with the 3-phosphoglycerate.