PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF SOYBEAN SEED ACID-PHOSPHATASES

Four isoforms of acid phosphatase (EC 3.1.3.2), API, AP2, AP3A and AP3 B, have been detected and partially purified from soybean seed (Glycin e max) through DEAE- and SP-Sephadex chromatographies. Specific activi ty values of 822, 163, 14 and 66 nkat.mg(-1) were obtained for API (90 3-fold purification), AP2 (180-fold), AP3A (15-fold), and AP3B (73-fol d), respectively, using p-nitrophenylphosphate as substrate. Relative native molecular mass values for API, AP2, AP3A and AP3B, determined b y gel filtration on calibrated SW-300 Waters Protein Glass column, wer e found to be 51 000, 58 000, 52 000 and 30 000, respectively. All fou r acid phosphatase isoforms presented a carbohydrate moiety in their s tructures and revealed only single phosphatase activity bands followin g nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. AP1 and AP2 exhibited greater substrate specificity than AP3A and AP3B. The K -m values were determined for p-nitrophenylphosphate, tyrosine-phospha te and inorganic pyrophosphate, at pH 5.0 and 37 degrees C. The acid p hosphatases presented the following apparent K-m values: API (pNPP - 0 .49, PPi - 0.21 and TyrP - 1.14 mM); AP2 (pNPP - 0.38, PPi - 1.33 and TyrP - 1.14 mM); AP3A (pNPP1 - 0.20, PPi - 0.16 and TyrP - 0.19 mM) an d AP3B (pNPP - 0.086, PPI 0.17 and TyrP 0.17 mM). All four isoforms we re inhibited by inorganic phosphate, fluoride, vanadate, molybdate, Cu 2+ and Zn2+. The soybean seed acid phosphatases did not catalyze the t ransphosphorylation reaction since no stimulation was observed with in organic phosphate accepters, such as glycerol, methanol and ethanol. ( C) Elsevier, Paris.