Partial purification and characterization of phosphoenolpyruvate carboxylase from the cyanobacterium Anabaena variabilis

Abstract

The partial purification and characterization of phosphoenolpyruvate (PEP) carboxylase from the cyanobacterium Anabaena variabilis is reported. Spheroplasts made from photoautotrophically grown cells were lysed to produce a crude cell extract which was fractionated by (i) (NH4)2SO4 precipitation and (ii) gel filtration through Sephacryl S-300. The peak of enzyme activity that was eluted from the column corresponded to a molecular mass of approximately 365 000 daltons; the molecular mass of the sub-units was found to be approximately 100 500 daltons as assessed by polyacrylamide gel electrophoresis. The pH optimum for PEP carboxylase activity was pH 7.0. The response of this A. variabilis enzyme to effectors distinguished this enzyme from that found in C3 plants and in bacteria. Malate inhibited enzyme activity but was a much more effective inhibitor at pH 7.0 (50% inhibition at 1.6 mM malate) than at pH 8.0 (15% inhibition at 10 mM malate). Glycine stimulated PEP carboxylase activity at both pH 7.0 and 8.0, while glucose 6-phosphate had no significant effect. The Km for HCO3− was calculated to be 14.5 ± 9.2 μM. These results suggest that PEP carboxylase from A. variabilis is kinetically similar to maize PEP carboxylase and may participate in inorganic carbon uptake for photosynthesis.