Effect of Fe²⁺ and ascorbic acid on acid phosphatases from rat bone

Summary

The effect of ferrous iron (Fe²⁺) and the reducing agents cysteine, dithiothreitol, and especially ascorbate on acid phosphatase activity was determined in vitro. Activity was extracted from homogenates of suckling rat tibia and femur with hypertonic KCl and Triton X-100, treated with protamine to remove interfering macromolecules, and dialyzed. Tartrate-sensitive and tartrate-resistant activity were separated and partially purified by cation exchange chromatography. At optimum dosage levels, Fe²⁺ was 100 times more potent than reducing agents in stimulating activity. Hypertonic KCl facilitated the effects of all agents. Fe²⁺ had no effect on tartrate-sensitive activity (E₁), but specifically stimulated the hydrolysis ofβ-glycerophosphate, casein, and especially ATP andp-nitrophenylphosphate (p-NPP) by tartrate-resistant enzyme (E₂); other divalent cations were either inhibitory or ineffective over a concentration range of 10⁻⁵ to 10⁻²M. Stimulation of E₂ was detectable at 10⁻⁶M Fe²⁺, and the effect was synergistically increased by 10⁻³M ascorbate. E₂ was stimulated maximally at 10⁻⁴M Fe²⁺ + 10⁻³M ascorbate, but at these concentrations their combined effects were additive. Both stimulated and unstimulated enzyme had identical pH optima (5.8), but the activity of the stimulated enzyme declined more slowly at higher pH values. Hypertonic KCl, Fe²⁺, or ascorbate reduced one-fourth to one-half the K_m of activity withp-NPP substrate, which suggested a direct effect of these substances on E₂. It is postulated the Fe²⁺ may interact with sulfhydryl groups in E₂, and that reducing agents and KCl may facilitate this action by (a) maintaining the enzyme in an optimal conformational state, and (b) keeping iron reduced as Fe²⁺.