Purification, structure, and catalytic properties of L-myo-inositol-1-phosphate synthase from rat testis.

L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4) has been purified to homogeneity for the first time and its properties were investigated. By means of ammonium sulfate precipitation from rat testis supernatant, followed by chromatography on DEAE-cellulose, Ultrogel, glucose 6-phosphate-Sepharose, and hydroxylapatite, the synthase was purified 460-fold to a specific activity of 250 milliunits/mg of protein (4.2 mkat/kg). The enzyme requires NAD+ and is optimally active at pH 7.7. The molecular weight determined by chromatography, electrophoresis, and sedimentation equilibrium is 210,000; the subunit weight is 68,000, a result which suggests a rare trimeric structure. Monovalent cations, among which N€L’ is the most effective, decreased the K,,, for both glucose 6-phosphate and NAD’ from the basal values of 3.8 l l l ~ and 17.9 p ~ , respectively. With the exception of Li’, which is inhibitory, monovalent cations increased the activity up to 400%, suggesting a possible regulatory role for these ions. Divalent cations had no effect and heavy metals inhibited strongly. EDTA had no effect, suggesting that although metals can alter the activity, metal is not required for basal activity. 2-Deoxyglucose Q-phosphate and 2-deoxyglucitol 6-phosphate inhibited strongly. Antibody against the homogeneous enzyme, prepared in rabbits, cross-reacted with testis, epididymis, and brain synthases showing identity of the three enzymes. The immunoprecipitated enzyme complex from either testis or brain is about 60% as active as the corresponding soluble enzyme. No binding of labeled substrate through a Schiff base could be detected, nor was the enzyme inactivated by borohydride reduction. Recent studies showing that epididymis is a richer source of synthase than testis could not be confirmed. In testis of diabetic animals, although free inositol levels were 2 to 3 times higher than normal, there was no change in the level of inositol-1-phosphate synthase.