L-myo-Inositol-1-phosphate synthase has been purified to homogeneity from bovine testis by (NH4)2SO4 precipitation on Celite followed by reverse (NH4)2SO4 gradient elution, DEAE chromatography, gel filtration, and hydroxylapatite chromatography. The enzyme is then pure by the criteria of elution profile from the hydroxylapatite, electrophoresis, and sedimentation properties. We find no overall (gluconeogenic) reversibility of the enzyme using 6 mM DL-myo-inositol-1-P. The first three steps of the reaction are reversible as determined by uptake of isotope from a D2O incubation medium into the 6 position of D-glucose-6-P. Thus, substrate binding, dehydrogenation, and proton removal prior to the aldol cyclization are all reversible steps. The enzyme is less than 5% NAD+ independent and is not inhibited by substrate or product (5 mM D-glucose-6-P or 0.8 mM DL-myo-inositol-1-P). The enzyme is twofold stimulated by either 50 mM NH4+ or 50 mM K+; the activation by these ions is not additive. Sodium ions inhibit the enzyme by 78% at 153 mM. The effect of sodium and potassium is not on the Km of D-glucose-6-P but on Vmax. We propose that K+ activates the enzyme by stabilizing a carbanion intermediate. Ethanol stimulates the enzyme 2-fold and 2.5-fold with added K+. The effect of ethanol appears to be via lowering of the D-glucose-6-P Km. In the presence of ethanol the effect of salt on Vmax disappears.

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