Multiple forms of barley root acid phosphatase: purification and some characteristics of the major cytoplasmic isoenzyme

Abstract

The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electroporesis and moves as a single hand of M r ∼ 38 000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was M r 77 600 and 79 000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate ( K i = 9 · 10 −7 M). NaF, Ag +, Hg 2+, Pb 2+ and Zn 2+ are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4–8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.