Abstract
An assay is described for the characterization of inositol monophosphatase (EC 3.1.3.25, 1L-myo-inositol -1- phosphatase) in Epstein-Barr virus transformed human B-lymphocytes. Cells from 600 ml expansions of frozen cultures were lysed by homogenization at low ionic strength and the soluble enzyme enriched by ammonium sulfate precipitation. The presence of Mg2+ and reducing conditions as necessary for maximal recovery of activity. Enzyme preparations containing 10 to 100 μg of protein were assayed for kinetic parameters by incubation with Ins(1)P (L-myo-inositol 1-phosphate); and measurement of the product myo-inositol using GC/MS. The human enzyme was found to have characteristics similar to those of other mammalian inositol monophosphatases: K(m) Ins(1)P, 90 ± 5μM (mean ± SD); K(i) (Li+) 1.19 ± 0.18 mM; K(i) (Ca2+) 123 ± 66 μM; K(i) (Mn2+), 7.14 ± 0.47 μM. As with the other mammalian enzymes, lithium is an uncompetitive inhibitor of the phosphatase. As an example of the usefulness of the method, the kinetic parameters of inositol monophosphatase from five healthy individuals were measured and the K(i) of lithium was found to have substantial population variation.
| Original language | English |
|---|---|
| Pages (from-to) | 49-54 |
| Number of pages | 6 |
| Journal | Lithium |
| Volume | 3 |
| Issue number | 1 |
| State | Published - Jan 1 1992 |