Improved Purification and Further Characterization of Acetyl‐CoA Carboxylase from Cultured Cells of Parsley (Petroselinum hortense)

Abstract

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 avidin‐ monomer ‐ 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 K_m values for the substrates were: acetyl‐CoA, 0.15mmol/1; bicarbonate, 1 mmol/1; MgATP²⁻, 0.07mmol/1. The carboxylase was inhibited by > 50mmol/1 NaC1, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/1, 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.