The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short-term thermal stress of the alga.
The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn(2+) chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast-frozen cell extracts was stable at -80 degrees C and, after thawing and storage on ice, remained stable for up to 1 h before declining non-linearly. Crude cell extracts bound [(14)C] 2-carboxy-D-arabitinol 1,5-bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre-treatment of Symbiodinium cell cultures in darkness at temperatures above 30 degrees C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36 degrees C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.
Lilley
Ross McC
RM
School of Biological Sciences, University of Sydney, NSW 2008, Australia. ross.lilley@sydney.edu.au
Ralph
Peter J
PJ
Larkum
Anthony W D
AW
D020134:Catalytic Domain; D002457:Cell Extracts; D002478:Cells, Cultured; D004141:Dinoflagellida / Q000201:enzymology*; D004789:Enzyme Activation; D008163:Luminescent Measurements / Q000379:methods*; D008345:Manganese / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013312:Stress, Physiological; D013696:Temperature; D013997:Time Factors
D002457:Cell Extracts; D008345:Manganese; D012273:Ribulose-Bisphosphate Carboxylase