ISOLATION AND CHARACTERIZATION OF A TYROSYL PHOSPHATASE ACTIVATOR FROM RABBIT SKELETAL-MUSCLE AND XENOPUS-LAEVIS OOCYTES
ISOLATION AND CHARACTERIZATION OF A TYROSYL PHOSPHATASE ACTIVATOR FROM RABBIT SKELETAL-MUSCLE AND XENOPUS-LAEVIS OOCYTES
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DOI：
10.1021/bi00455a010
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发表时间：
1990-01-23
期刊：
影响因子：
2.9
通讯作者：
MERLEVEDE, W
中科院分区：
文献类型：
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作者：
CAYLA, X;GORIS, J;MERLEVEDE, W
PTPA是一种PCSH2和PCSL蛋白磷酸酶的特异性磷酸化酪氨酸磷酸酶激活剂，从非洲爪哇卵巢和兔骨骼肌中分离纯化，并从狗肝脏中高度纯化。在凝胶过滤和蔗糖梯度离心法中，PTPA以40 kDa的蛋白形式出现，而在SDS-PAGE中则以37-39 kDa的蛋白双链形式出现。估计其在卵母细胞中的细胞浓度为0.75µm，在兔骨骼肌中的浓度为0.25µm，这表明它在细胞PTPase活性的调节中发挥了重要作用。PCSL磷酸酶的PTPase激活反应是时间依赖的，ATP和Mg2+是必需的辅因子[在5 mM的MgCl2存在下，A50(ATP)=0.12 mM]。以RCM溶菌酶为底物，PTPA激活的PCSL磷酸酶的比活力为700nmolPI/(min.cntdot.mg)。PTPase的最适pH由基础条件的8.5-9转变为中性pH(7-7.5)，必需金属离子镁的A50降低了3 mM。在底物存在的情况下，激活迅速逆转，在去除ATP.cntdot.MG后，激活速度更慢。PTPA激活的PCSL磷酸酶是乳突圆线虫卵母细胞胞浆中的主要PTPase活性(至少有50%的可测PTPase与RCM溶菌酶在酪氨酸残基上发生磷酸化)。PTP的激活专用于PCSL和PCSH2磷酸酶的PTPase活性，而不影响其磷酸丝氨酸/苏氨酰磷酸酶的活性。然而，磷酸化酶磷酸酶活性的影响因素，如多阳离子和冈田酸，也影响PTPase活性。磷酸化酶a抑制激活的PTPase活性(I50=5微米)。其他寡聚体PCS磷酸酶(PCSH1和PCSM)的PTPase不受影响，这表明它们的一些亚基具有抑制作用。将这种激活与最近描述的由ATP/PPI[Goris，J.，Pallen，C.J.，Parker，P.J.，Hermann，J.，Watfield，M.D.和Merleveed，W.(1988)Biochem，J.256,1029-1034]和微管蛋白[Jessus，C.，Goris，J.，Cayla，X.，Hermann，J.，Hendrix，P.，Ozon，R.和Merleveed，W.]对PCS磷酸酶的激活进行比较。
PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 .mu.M in oocytes or 0.25 .mu.M in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.cntdot.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.cntdot.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTP activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase a inhibits the activated PTPase activity (I50 = 5 .mu.M). The PTPase of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C.J., Parker, P.J., Hermann, J., Waterfield, M.D., and Merlevede, W. (1988) Biochem, J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., and Merlevede, W.