Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli.
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.
Park
Sang-Hee
SH
Department of Food and Microbial Technology, Seoul Women's University 126 Kongnung 2-dong, Nowon-Gu, Seoul 139-774, Korea.
Kim
Jong-Il
JI
D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D007295:Inositol Phosphates; D021122:Protein Subunits; D011994:Recombinant Proteins; D009243:NAD; C002647:inositol 1-phosphate; D019298:Glucose-6-Phosphate; D007296:Myo-Inositol-1-Phosphate Synthase