Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli

J Microbiol. 2004 Mar;42(1):20-4.

Abstract

Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C. The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000. A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, Gel
  • Cloning, Molecular
  • Drosophila melanogaster / enzymology*
  • Drosophila melanogaster / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Glucose-6-Phosphate / metabolism
  • Hydrogen-Ion Concentration
  • Inositol Phosphates / metabolism
  • Kinetics
  • Molecular Weight
  • Myo-Inositol-1-Phosphate Synthase / chemistry
  • Myo-Inositol-1-Phosphate Synthase / genetics*
  • Myo-Inositol-1-Phosphate Synthase / isolation & purification
  • Myo-Inositol-1-Phosphate Synthase / metabolism
  • NAD / metabolism
  • Protein Subunits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Temperature

Substances

  • Inositol Phosphates
  • Protein Subunits
  • Recombinant Proteins
  • NAD
  • inositol 1-phosphate
  • Glucose-6-Phosphate
  • Myo-Inositol-1-Phosphate Synthase