Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was pu- rified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molec- ular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE (under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands, pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phos- phate was estimated to be 0.16 mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The en- zyme is inhibited competitively by phosphate (K i = 1.25 mM), molybdate (K i = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg 2+ ,C u 2+ and Zn 2+ express an inhibitory effect on the enzyme, while the en- zyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.