Complete purification and general characterization of FAD synthetase from rat liver

J Biol Chem. 1987 May 25;262(15):7418-22.


Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.

Publication types

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

MeSH terms

  • Animals
  • Cations, Divalent
  • Chromatography
  • Drug Stability
  • Electrophoresis, Polyacrylamide Gel
  • Flavin-Adenine Dinucleotide / biosynthesis
  • Fractional Precipitation
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Liver / enzymology*
  • Macromolecular Substances
  • Magnesium / pharmacology
  • Molecular Weight
  • Nucleotidyltransferases / isolation & purification*
  • Nucleotidyltransferases / metabolism
  • Rats


  • Cations, Divalent
  • Macromolecular Substances
  • Flavin-Adenine Dinucleotide
  • Nucleotidyltransferases
  • FMN adenylyltransferase
  • Magnesium