Bakalara N, Seyfang A, Davis C, Baltz T
Laboratorie d'Immunologie et Parasitologie Moléculaire, Université Bordeaux II, France.
Eur J Biochem. 1995 Dec 15;234(3):871-7. doi: 10.1111/j.1432-1033.1995.871_a.x.
We report the first characterization of plasma-membrane-bound tyrosine phosphatase activity in the haemoprotozoan. Trypanosoma brucei. Several enzymic properties of the membrane fraction were identical to other protein tyrosine phosphatases (PTPases), such as (a) insensitivity to inhibitors of other protein phosphatases, including tetramisole, sodium tartrate and okadaic acid, (b) inhibition by sodium vanadate, and (c) activation by spermidine. Additionally, T. brucei PTPase activity presented two novel features, an acidic pH optimum at pH 4.0-5.0 and a very low Km value (2.5 nM) for the specific synthetic substrate, Tyr(P)Raytide. Higher Km values of 170 nM for Tyr(P)-RCML (RCML, reduced, carboxamidomethylated and maleylated lysozyme) and of 3 mM for the non-specific inorganic substrate p-nitrophenyl phosphate, suggested that the PTPase activity of T. brucei was substrate specific. Reconstitution experiments on bloodstream-stage membrane proteins revealed that three polypeptides of 148, 115 and 72 kDa contained vanadate-inhibitable PTPase activity. Modulator assays revealed that the 72-kDa protein was responsible for the observed spermidine stimulation, but indicated that the modulator profile of the 148-kDa protein was most similar to the whole membrane fraction. Furthermore, the PTPase activity of T. brucei was life-cycle-stage regulated. Neither the whole membrane fraction nor the reconstituted proteins of the procyclic insect stage dephosphorylated tyrosine residues.
我们报道了血原生动物布氏锥虫中质膜结合酪氨酸磷酸酶活性的首次表征。膜部分的几种酶学特性与其他蛋白酪氨酸磷酸酶（PTPases）相同，例如：（a）对包括四咪唑、酒石酸钠和冈田酸在内的其他蛋白磷酸酶抑制剂不敏感；（b）被钒酸钠抑制；（c）被亚精胺激活。此外，布氏锥虫PTPase活性呈现出两个新特征，最适酸性pH为4.0 - 5.0，对特异性合成底物Tyr(P)Raytide的Km值非常低（2.5 nM）。对于Tyr(P)-RCML（RCML，还原、羧酰胺甲基化和马来酰化溶菌酶）的Km值较高，为170 nM，对于非特异性无机底物对硝基苯磷酸酯的Km值为3 mM，这表明布氏锥虫的PTPase活性具有底物特异性。对血流期膜蛋白的重组实验表明，148、115和72 kDa的三种多肽具有钒酸盐抑制的PTPase活性。调节剂分析表明，72 kDa的蛋白负责观察到的亚精胺刺激，但表明148 kDa蛋白的调节剂谱与整个膜部分最相似。此外，布氏锥虫的PTPase活性受生命周期阶段调节。前循环昆虫阶段的整个膜部分或重组蛋白都不能使酪氨酸残基去磷酸化。
