Potential contribution of β-carboxylases to photosynthetic carbon isotope fractionation in a marine diatom

In vitro activities of phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) were measured in chemostat cultures of the marine diatom Phaeodactylum tricornutum grown under either phosphate- or nitrate-limited conditions and at low (I [mu mol kg(-1)) and high (70-100 mu mol kg(-1)) CO2 concentrations. A comparison of in vitro methods for measuring PEPC activity underscored the importance of including glycerol in the reaction mixture because of its ability to stabilize the quaternary structure of PEPC. Assays for total P-carboxylase activity (PEPC + PEPCK) identified the importance of substituting Mn2+ for Mg2+ as an activator for PEPC since Mg2+ is inhibitory to PEPCK activity. Results indicated that PEPC was constitutive, that is, present in a constant ratio relative to the limiting nutrient over a wide range of growth conditions. PEPCK acclimation was greater, and its intracellular concentration was positively correlated with CO2 concentrations. In vitro P-carboxylase activity averaged 13% of in vivo carbon fixation rates. The low percentage of beta-carboxylase activity and the positive correlation between PEPCK and CO2 concentration argue against a C-4-type pathway in P. tricornutum; that is, P-carboxylase activity appears related to strictly anaplerotic processes. Calculations indicate that P-carboxylase activity reduces the fractionation associated with carbon fixation but by no more than 2%.