Untersuchungen zur Rolle der Phosphoenolpyruvat-Carboxykinase und des NADP+-Malat-Enzyms in der filamentösen Phaeophycee Ectocarpus siliculosus

The filamentous brown-alga Ectocarpus siliculosus shows high activities of phosphoenolpyruvate carboxykinase (PEPCK) and NADP+-malic enzyme. The putative participation of both enzymes in a C4-like carbon cycle became questionable because not all enzymes necessary for this cycle could be detected and because the relevant metabolite pools in vivo were too small. This raised the question of their cellular function. In order to clarify their function, PEPCK and the NADP+-malic enzyme were purified to homogeneity. The molecular mass of native PEPCK was determined to be 90 kDa. SDS-PAGE revealed two polypeptides of 62 kDa and 18 kDa, respectively. The molecular mass of native malic enzyme was 440 kDa. SDS-PAGE revealed only one polypeptide of 56 kDa which suggested that the native molecule might be a homo-octamer. The carboxylating-reaction of PEPCK displayed typical Michaelis-Menten-Kinetics for each substrate. The Km was 1,46 mM for CO2, 0,5 mM for PEP and 0,23 mM for ADP. The double-reciprocal plot of the activity of NADP+-malic enzyme as a function of the concentration of L-malate yielded a straight line with a Km for L-malate of 0,35 mM. With respect to the binding of NADP+ malic enzyme revealed positive cooperativity with a Hill coefficient of 2,74. This indicates at least three binding sites for NADP+. Rabbits were immunised using the denatured polypeptides after SDS-PAGE. Specific antibodies were obtained only against the 18 kDa polypeptide of PEPCK. In immunoblots, the antibodies reacted with both, the 18 kDa and the 62 kDa polypeptide of the PEPCK, indicating that they were both degradation products from a single probably monomeric polypeptide (the native 90 kDa protein). In immuno-localisation studies this antibody against PEPCK stained the pyrenoids of E. siliculosus. With antibodies against native Rubisco from spinach, Rubisco was also found to be exclusively localised in the pyrenoids. The localisation of PEPCK in direct neighbourhood to Rubisco is so far the strongest argument against the existence of a C4 pathway in E. siliculosus. An anaplerotic role of PEPCK in the alga is likely.