Purification and Characterization of Phosphoenolpyruvate Carboxylase from Plasmodium berghei

Abstract

Phosphoenolpyruvate (PEP) carboxylase was purified over 400-fold from Plasmodium berghei . The purified enzyme was stable in 0.4 m potassium phosphate buffer ( p H 7.4) containing 0.5 m glucose, 1 m m ethylenediaminetetraacetic acid (EDTA), and 1 m m MgCl 2 . It had a molecular weight of 280,000 determined by sucrose density gradient centrifugation in this buffer, but it aggregated and was unstable in the presence of different salts or a more dilute solution of potassium phosphate. The K m for PEP was 2.6 m m and that for Mg 2+ was 1.3 m m . The K m for bicarbonate was 2 m m . Citrate, nucleotides, and EDTA inhibited the PEP carboxylase of P. berghei by decreasing the concentration of free magnesium ions, but acetyl-coenzyme A, fructose-1,6-diphosphate, and aspartate did not influence its activity. A chloroquine concentration of 1.8 × 10 −4 m inhibited the enzyme 50%.