Purification and characterization of 3-methylcrotonyl-CoA carboxylase from somatic embryos of Daucus carota

Arch Biochem Biophys. 1993 Aug 15;305(1):103-9. doi: 10.1006/abbi.1993.1398.

Abstract

3-Methylcrotonyl-CoA carboxylase, a biotin enzyme, was purified from embryos of Daucus carota. Polyethylene glycol precipitation and monomeric avidin affinity chromatography were used to purify all biotin enzymes from cell-free extracts of embryos. The resulting 3-methylcrotonyl-CoA carboxylase preparation had a specific activity of 745 nmol/min.mg protein, representing a 3725-fold purification of the enzyme and a 135% recovery of activity. Fractionation of the purified biotin-containing proteins by anionic exchange chromatography using Q-Sepharose partially resolved the 3-methylcrotonyl-CoA carboxylase from the other biotin enzymes. 3-Methylcrotonyl-CoA carboxylase has a biotin-containing subunit with a molecular mass of about 78,000 Da and a non-biotin-containing subunit of about 65,000 Da. The native enzyme is 987,000 Da. The optimum pH for activity is between 8.0 and 8.4. The apparent Km values for the substrates 3-methylcrotonyl-CoA, sodium bicarbonate, and ATP are 42 +/- 2 microM, 4.0 +/- 0.9 mM, and 21 +/- 2 microM, respectively. The enzyme is inhibited by acetoacetyl-CoA and palmitoyl-CoA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acyl Coenzyme A / metabolism
  • Adenosine Triphosphate / metabolism
  • Bicarbonates / metabolism
  • Bicarbonates / pharmacology
  • Biotin / analysis
  • Carbon-Carbon Ligases*
  • Chemical Fractionation
  • Chemical Precipitation
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Hydrogen-Ion Concentration
  • Kinetics
  • Ligases / chemistry
  • Ligases / isolation & purification*
  • Ligases / metabolism
  • Molecular Weight
  • Polyethylene Glycols
  • Seeds / enzymology*
  • Sodium / metabolism
  • Sodium / pharmacology
  • Sodium Bicarbonate
  • Substrate Specificity
  • Vegetables

Substances

  • Acyl Coenzyme A
  • Bicarbonates
  • Polyethylene Glycols
  • tiglyl-coenzyme A
  • Biotin
  • Adenosine Triphosphate
  • Sodium Bicarbonate
  • Sodium
  • Ligases
  • Carbon-Carbon Ligases
  • methylcrotonoyl-CoA carboxylase