Improved purification and further characterization of acetyl-CoA carboxylase from cultured cells of parsley (Petroselinum hortense).
Acetyl-CoA carboxylase from irradiated cell-suspension cultures of parsley (Petroselinum hortense) has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidinmonomer--Sepharose 4B. Molecular weights of about 420000 for the native enzyme and about 220000 for the enzyme subunit were determined respectively by gel filtration or sucrose-density-gradient sedimentation and by electrophoresis in the presence of dodecyl sulfate. The purified enzyme showed an isoelectric point of 5. The enzyme carboxylated the straight-chain acyl-CoA esters of acetate, propionate, and butyrate at decreasing rates in this order. The catalytic efficiency of the carboxylase was highest when ATP existed largely as MgATP2- complex. At the optimum pH of 8 the apparent Km values for the substrates were: acetyl-CoA, 0.15 mmol/1; bicarbonate, 1 mmol/1; MgATP2-, 0.07 mmol/1. The carboxylase was inhibited by greater than 50 mmol/l NaCl, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/l, but strongly inhibited the carboxylase at higher concentrations. The results of these studies demonstrate that several properties of the light-inducible acetyl-CoA carboxylase of parsley cells, an enzyme of the flavonoid pathway, are remarkably similar to those of acetyl-CoA carboxylases from a variety of other organisms.
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification* / Q000378:metabolism; D001710:Biotin / Q000032:analysis; D002384:Catalysis; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D008025:Ligases / Q000302:isolation & purification*; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification*
D010940:Plant Proteins; D001710:Biotin; D008025:Ligases; D000103:Acetyl-CoA Carboxylase