Enzymes associated with β-carboxylation in Ectocarpus siliculosus (Phaeophyceae): Are they involved in net carbon acquisition ?

Abstract

The hypothesis that carbon acquisition in phaeophytes of lower organization is controlled by a blue-light-regulated C₄ mechanism was tested by various approaches with the filamentous species Ectocarpus siliculosus. Here we report analyses at the enzymatic level. Enzymes potentially participating in a C₄ cycle were assayed in extracts, and activities of a phosphoenolpyruvate carboxykinase (PEPCK), of NAD- and NADP-malate dehydrogenases, and of a NADP-malic enzyme were detected. No activity could be measured of phosphoenolpyruvate carboxylase or of pyruvate-Pi-dikinase, which is essential to complete the cycle by regenerating phosphoenolpyruvate from pyruvate. The kinetic properties of all enzymes were determined. Malic enzyme and PEPCK were purified to homogeneity, as tested by gel filtration. The molecular mass of native malic enzyme was determined to be 440 kDa. In SDS gel electrophoreses it produced a single band with a relative molecular mass of 53 kDa, which suggested that the native molecule might be a homo-octamer. The molecular mass of PEPCK was 90 kDa and separation on SDS gels produced two major bands, one of 56 kDa and one of 18 kDa, which appeared not to be a degradation product of the larger polypeptide. The composition of the native enzyme is unclear. In extracts, the activities of malic enzyme and PEPCK followed a circadian rhythm, but none of the enzymes tested was found to be blue-light-dependent. The above results neither prove nor dismiss the possibility that a C₄ mechanism operates in Ectocarpus.