Comparison of Phosphoenolpyruvate-Carboxykinase from Autotrophically and Heterotrophically Grown Euglena and Its Role during Dark Anaerobiosis

Abstract

Eugkna grailis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO2 in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO2 alone and kept in the dark, whereas highest yields result if both glucose and CO2 are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions.

The role of phosphoenolpyruvate carboxykinase in a marine macroalga with C4-like photosynthetic characteristics.

“…A decarboxylation role for PEPCK is supported by the fact that malate-driven photosynthesis is almost 70%o lower in the presence of MPA. The PEPCK of higher plants is located in the cytoplasm (5,22), although in other organisms it has more than one intracellular location (7)(8)(9). We are attempting to establish the location(s) of PEPCK in Udotea cells.…”

“…However, many marine algae show no 02 inhibition of photosynthesis (1)(2)(3) (6), while cytosolic and mitochondrial isozymes, acting as GTPrequiring decarboxylases, regulate mammalian gluconeogenesis (7,8). In the algal protist Euglena, two forms operate: one as a gluconeogenic decarboxylase and one in CO2 fixation for the methylmalonyl CoA pathway (9). Substantial C4 acid production by the B-carboxylation of phosphoenolpyruvate occurs in some marine algae (1,2).…”

Variations in carboxylase activity in marine phytoplankton cultures, ß-carbolxylation in carbon flux studies

“…Strict heterotrophic species may incorporate inorganic carbon (Levedahl 1968, Raven 1974. In heterotrophically grown Euglena gracilis, inorganic carbon assimilation occurred through PEPCK activity (Briand et al 1981, Peak & Peak 1981, Ponsgen-Schmidt et al 1988). However, for heterotrophic cells, information is lacking about the origin of phosphoenolpyruvate.…”

Euglena gracilis Rhodoquinone:Ubiquinone Ratio and Mitochondrial Proteome Differ under Aerobic and Anaerobic Conditions

“…Rhodoquinone Levels in Euglena Change with Oxygen Availability-In wax ester fermentation in Euglena mitochondria, the synthesis of even-numbered fatty acids starts from acetylCoA, whereas odd-numbered fatty acid synthesis starts from propionyl-CoA, which is generated via succinate, propionate, and methylmalonyl-CoA (21,56,57). The formation of propionyl-CoA involves fumarate reductase, which catalyzes the reverse reaction of that in succinate dehydrogenase (complex II) but requires the lower midpoint potential of rhodoquinone versus ubiquinone to function efficiently in the succinate-synthesizing direction (2,13,58,59).…”