Extraction, Partial Purification and Characterization of Phosphoenolpyruvate Carboxykinase From Ascophyllium Nodosum (Phaeophyceae)¹

Phosphoenolpyruvate carboxykinase (PEPCK) from Ascophyllum nodosum (L.) Le Jolis was partially purified and characterized to investigate its role in inorganic carbon assimilation in macroalgae. Inorganic carbon isotopic disequilibrium studies showed that the carboxylation of phosphoenolpyruvate utilized CO 2 rather than HCO 3 − as its source of inorganic carbon. This is consistent with the enzyme being a phosphoenolpyruvate carboxykinase rather than a phosphoenolpyruvate carboxylase. Pre‐incubation with Mn 2+ alone activated PEPCK more effectively than when combinations of Mn 2+ , ADP and HCO 3 − were used as activators. Activation of PEPCK during catalysis was found not to occur. Although the activation of PEPCK reduced the K m for CO 2 by a factor of 2.25, the value reported here of 1.084 mM CO 2 for the activated enzyme at pH 7.0 is at the top of the range of previously reported values for brown algal PEPCK. The specific activity of PEPCK was increased from 0.268 μmol·min −1 ·mg −1 in the crude extract to 33.03 μmol·min −1 ·mg −1 in the partially purified preparations. Whether PEPCK can act as an initial carboxylating enzyme is discussed. Triton X‐100 at 0.57% (v/v) was found to be the optimum detergent and concentration for the extraction of enzymes from A. nodosum. When high concentrations of detergents ‐were used, a low (NH 4 ) 2 SO 4 cut was required to remove the free detergent from solution, which was extracted by centrifugation. Q Sepharose was used to partially purify PEPCK and separate it from pyruvate kinase. Good protein separations were consistently obtained.