Phosphatase activities, determined mainly by the hydrolysis of p-nitrophenyl phosphate (p-NPP), were found in porcine brain microtubule proteins prepared by successive cycles of assembly and disassembly. The phosphatase activity showed a pH-optimum around 5 with p-NPP as a substrate in the presence of ethylenediaminetetraacetic acid or Mg2+. The addition of Zn2+ suppressed acid and neutral phosphatase activities, but enhanced the activities 5-70 folds over pH 9. The phosphatase activity contained in microtubule proteins showed low substrate specificity. Fructose-1, 6-diphosphate was hydrolyzed more rapidly than p-NPP and fructose-6-phosphate. Furthermore, the enzyme(s) catalyzed the hydrolysis of phospho-D, L-tyrosine, but rarely acted on phospho-D, L-threonine and phospho-L-serine. The addition of ammonium molybdate which considerably inhibits the hydrolysis of p-NPP, showed a slight effect on phosphatase activity toward casein or phosvitin as substrates. The addition of ascorbic acid enhanced the hydrolysis of p-NPP, whereas it rather suppressed the phosphatase activity against casein and phosvitin. Ammonium molybdate and ascorbic acid did not affect microtubule polymerization. When microtubule proteins were separated into tubulin and microtubule-associated proteins (MAPs) by phosphocellulose column chromatography, the activities at pH 5, 7, and 10 were mostly recovered in 0-0.8 M KCl eluted fractions, indicating that total phosphatases are contained in MAPs, not in tubulin. The addition of tubulin, actin, and calmodulin which are capable of binding to MAPs, had little effect on the activity.
