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

The hypothesis that carbon acquisition in phaeophytes of lower organization is controlled by a blue-light-regulated C-4 mechanism was tested by variou s approaches with the filamentous species Ectocarpus siliculosus. Here we r eport analyses at the enzymatic level. Enzymes potentially participating in a C-4 cycle were assayed in extracts, and activities of a phosphoenolpyruv ate carboxykinase (PEPCK), of NAD- and NADP-malate dehydrogenases, and of a NADP-malic enzyme were detected. No activity could be measured of phosphoe nolpyruvate carboxylase or of pyruvate-Pi-dikinase, which is essential to c omplete the cycle by regenerating phosphoenolpyruvate from pyruvate. The ki netic properties of all enzymes were determined Malic enzyme and PEPCK were purified to homogeneity, as tested by gel filtration. The molecular mass o f native malic enzyme was determined to be 440 kDa. In SDS gel electrophore ses it produced a single band with a relative molecular mass of 53 kDa, whi ch suggested that the native molecule might be a homo-octamer. The molecula r mass of PEPCK was 90 kDa and separation on SDS gels produced two major ba nds, one of 56 kDa and one of 18 kDa, which appeared not to be a degradatio n product of the larger polypeptide. The composition of the native enzyme i s 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-ligh t-dependent. The above results neither prove nor dismiss the possibility th at a C-4 mechanism operates in Ectocarpus.