title: A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.
abstract: Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.
authors: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
authors: Xinyi
authors: X
authors: Huang
authors: Marcy
authors: M
authors: Hernick
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D007296:Myo-Inositol-1-Phosphate Synthase / Q000032:analysis* / Q000378:metabolism*; D010504:Periodic Acid / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*; D015032:Zinc / Q000378:metabolism
chemical_list: D010504:Periodic Acid; D008345:Manganese; C009288:metaperiodate; D007296:Myo-Inositol-1-Phosphate Synthase; D008274:Magnesium; D015032:Zinc