The separation method and some properties of isodynamic acid phosphatases catalyzing FMN hydrolysis in spinach leaves were investigated.
1. The enzyme catalyzing FMN hydrolysis was found to occur practically and exclusively in the final supernatant fraction by differential centrifugation.
2. Acid phosphatase catalyzing FMN hydrolysis was separated into three fractions by chromatography on Sephadex G-100, equilibrated with 0.2M glycine buffer (pH 9.0).
3. pH optima of the three fractions were similarly 5.0.
4. Each affinity of the three enzymes for FMN (Km value) were calculated to be 1.8×10^-4 (Fraction I), 1.2×10^-4 (Fraction II) and 7.0×10^-4M (Fration III), respectively.
5. The substrate specificity for the three acid phosphatases was tested, and it was found that Fraction III was comparatively specific for FMN.
6. Fractions I and II were inhibited by ATP, ADP, orthophosphate and pyrophosphate, whereas Fraction III was inhibited by AMP and adenosine.