title: The activity of Rubisco's molecular chaperone, Rubisco activase, in leaf extracts.
abstract: Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the "molecular chiropractic" activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9-14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton, and wheat. Faster rates were observed in a transgenic line of Arabidopsis that expresses only the β-isoform of activase, whereas no activity was detected in a line that expresses only the α-isoform. Activase activity was also low or undetectable in rice, maize, and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco, and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.
authors: U.S. Department of Agriculture, Agricultural Research Service, Arid-Land Agricultural Research Center, 21881 N Cardon Lane, Maricopa, AZ 85138, USA. elizabete.carmo-silva@ars.usda.gov
authors: A Elizabete
authors: AE
authors: Carmo-Silva
authors: Michael E
authors: ME
authors: Salvucci
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays; D018869:Heat-Shock Response; D007527:Isoenzymes / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology* / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D018832:Molecular Chaperones; D010936:Plant Extracts; D010940:Plant Proteins; C054765:rca protein, plant; D012273:Ribulose-Bisphosphate Carboxylase