Pugazhenthi S, Tanha F, Dahl B, Khandelwal R L
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Arch Biochem Biophys. 1996 Nov 15;335(2):273-82. doi: 10.1006/abbi.1996.0508.
Inhibition of protein tyrosine phosphatase (PTP) activities by vanadate was examined in cultured rat hepatocytes. The incubation of hepatocytes with sodium orthovanadate inhibited PTP activities, measured with labeled polyglutamate tyrosine (4:1) and insulin receptor peptide (1142-1153), in a dose- and time-dependent manner. The PTP activities in cytosolic and particulate fractions were inhibited with the IC50 values of 30-50 and 2-20 microM, respectively. Vanadate-mediated inhibition of protein phosphatase, type 1 (a serine phosphatase) was less pronounced, requiring 50- to 150-fold higher concentrations. Molybdate and tungstate, the other potent inhibitors of PTPs, exerted approximately 70% less inhibition of enzyme activities compared to vanadate in intact liver cells. The cytosolic and particulate PTPs inhibited by vanadate were further resolved by fast protein liquid chromatography on Mono Q and Superose-12 columns. Vanadate exerted stable and differential inhibition of several PTPs. One of them was identified as SHPTP2 (Syp, SHP-2) in cytosolic as well as particulate fractions. Immunoprecipitation of this PTP with Syp-antibody coupled to protein A-agarose confirmed the vanadate-induced decrease in SHPTP2 activity. Vanadate did not alter the expression of SHPTP2 and its distribution between cytosolic and particulate fractions as indicated by the immunoblots. The decrease in the activities of PTPs in vanadate-treated hepatocytes in general was found to be reversed by the reducing agent dithioerythreitol. This study shows that vanadate inhibits many PTPs in intact liver cells, one of them being SHPTP2/SHP-2. The inhibition is stable after chromatography on ion-exchange and gel filtration chromatography. The enzyme inhibition seems to involve the oxidation of the thiol group of PTPs.
在培养的大鼠肝细胞中检测了钒酸盐对蛋白酪氨酸磷酸酶（PTP）活性的抑制作用。用原钒酸钠孵育肝细胞，以标记的聚谷氨酸酪氨酸（4:1）和胰岛素受体肽（1142 - 1153）测量，钒酸盐对PTP活性的抑制呈剂量和时间依赖性。胞质和微粒体部分的PTP活性受到抑制，IC50值分别为30 - 50 μM和2 - 20 μM。钒酸盐对1型蛋白磷酸酶（一种丝氨酸磷酸酶）的抑制作用不太明显，所需浓度高50至150倍。钼酸盐和钨酸盐是PTP的其他强效抑制剂，与钒酸盐相比，在完整肝细胞中对酶活性的抑制作用约低70%。通过在Mono Q和Superose - 12柱上进行快速蛋白质液相色谱进一步分离了被钒酸盐抑制的胞质和微粒体PTP。钒酸盐对几种PTP产生稳定且有差异的抑制作用。其中一种在胞质和微粒体部分均被鉴定为SHPTP2（Syp，SHP - 2）。用与蛋白A - 琼脂糖偶联的Syp抗体对该PTP进行免疫沉淀，证实了钒酸盐诱导的SHPTP2活性降低。免疫印迹表明，钒酸盐未改变SHPTP2的表达及其在胞质和微粒体部分之间的分布。一般发现，用还原剂二硫苏糖醇可逆转钒酸盐处理的肝细胞中PTP活性的降低。这项研究表明，钒酸盐抑制完整肝细胞中的许多PTP，其中之一是SHPTP2/SHP - 2。在离子交换和凝胶过滤色谱上进行色谱分离后，这种抑制作用是稳定的。酶抑制作用似乎涉及PTP硫醇基团的氧化。
