Purification and characterization of phosphoenolpyruvate carboxylase from Plasmodium berghei.
Phosphoenolpyruvate (PEP) carboxylase was purified over 400-fold from Plasmodium berghei. The purified enzyme was stable in 0.4 m potassium phosphate buffer (pH 7.4) containing 0.5 m glucose, 1 mm ethylenediaminetetraacetic acid (EDTA), and 1 mm 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 mm and that for Mg(2+) was 1.3 mm. The K(m) for bicarbonate was 2 mm. 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 x 10(-4)m inhibited the enzyme 50%.
D000645:Ammonium Sulfate; D001224:Aspartic Acid / Q000494:pharmacology; D002021:Buffers; D002247:Carbon Isotopes; D002254:Carbonates / Q000378:metabolism; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002951:Citrates / Q000494:pharmacology; D003065:Coenzyme A / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004589:Electrophoresis, Disc; D005374:Filtration; D005636:Fructosephosphates / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008274:Magnesium; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D010071:Oxaloacetates / Q000494:pharmacology; D010710:Phosphates; D010728:Phosphoenolpyruvate; D010961:Plasmodium / Q000201:enzymology* / Q000378:metabolism; D013053:Spectrophotometry; D013386:Succinates / Q000494:pharmacology; D013395:Sucrose
D002021:Buffers; D002247:Carbon Isotopes; D002254:Carbonates; D002951:Citrates; D005636:Fructosephosphates; D007656:Ketoglutaric Acids; D009711:Nucleotides; D010071:Oxaloacetates; D010710:Phosphates; D013386:Succinates; D001224:Aspartic Acid; D013395:Sucrose; D010728:Phosphoenolpyruvate; D004492:Edetic Acid; D002262:Carboxy-Lyases; D008274:Magnesium; D005947:Glucose; D003065:Coenzyme A; D000645:Ammonium Sulfate