Sopina V A
Institute of Cytology RAS, St. Petersburg.
Tsitologiia. 2002;44(11):1120-8.
Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.
在用2-萘基磷酸酯作为底物、pH值为4.0的聚丙烯酰胺凝胶圆盘电泳后显示的6条带（即电变体）中，变形虫（菌株B）的抗酒石酸酸性磷酸酶（TRAP）由其中3条带代表。用于凝胶染色的孵育混合物中存在MgCl2、CaCl2或ZnCl2（50 mM）时，会刺激所有3种TRAP电变体或其中两种（在ZnCl2的情况下）的活性。当凝胶在染色前用MgCl2、CaCl2或ZnCl2（10和100 mM，30分钟）处理时，TRAP电变体的活性也会增加。但与1 M MgCl2或1 M CaCl2不同，1 M ZnCl2会使三种TRAP电变体中的两种部分失活。EDTA和EGTA（5 mM）以及H2O2（10 mM）分别在凝胶处理10、20和30分钟后完全抑制TRAP电变体。在5种测试离子（Mg2+、Ca2+、Fe2+、Fe3+和Zn2+）中，只有后者能使先前因EDTA或EGTA处理而失活的TRAP电变体重新激活。此外，在EDTA失活后，TRAP电变体的重新激活效果比EGTA失活后更好。TRAP电变体对不同浓度的冈田酸和磷酸酶抑制剂混合物1的抗性表明这些电变体中不存在PP1和PP2A。TRAP活性对Mg2+、Ca2+和Zn2+的依赖性以及其电变体对钒酸盐和磷酸酶抑制剂混合物2的抗性阻止了这些电变体被归类为PTP。有人提出，变形虫TRAP的活性中心含有锌离子，这对该酶的催化活性至关重要。因此，这些变形虫的TRAP是一种金属磷酸酶，在酸性介质中表现出磷酸单酯酶活性。这种金属酶不同于哺乳动物的抗酒石酸PAPs和食用蛙的抗酒石酸金属磷酸酶。
