Colas B, Cambillau C, Buscail L, Zeggari M, Esteve J P, Lautre V, Thomas F, Vaysse N, Susini C
Institut National de la Santé et de la Recherche Médicale U 151, CHU Rangueil, Toulouse, France.
Eur J Biochem. 1992 Aug 1;207(3):1017-24. doi: 10.1111/j.1432-1033.1992.tb17138.x.
A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 microM and an apparent Vmax of 0.9 nmol.mg protein-1.min-1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31-56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1-1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [125I-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.
利用32P标记的聚（谷氨酸，酪氨酸）作为底物，已对大鼠胰腺腺泡细胞膜中的一种磷酸化蛋白酪氨酸磷酸酶（PTPase）活性进行了表征。腺泡细胞膜对该底物表现出高亲和力，表观Km为0.46微摩尔，表观Vmax为0.9纳摩尔·毫克蛋白-1·分钟-1。腺泡细胞膜PTPase活性表现出其他PTPase的特定特征；它受到抑制剂Zn2+、原钒酸盐以及二价阳离子Mn2+和Mg2+的抑制，并受到还原剂二硫苏糖醇的刺激。它也受到大豆胰蛋白酶抑制剂的抑制并受到胰蛋白酶的刺激。胰腺腺泡细胞膜的凝胶渗透产生了一个单一的酶活性峰，表观分子量为70000道尔顿。通过DEAE上的HPLC进一步纯化显示在120毫摩尔和180毫摩尔氯化钠处有两个PTPase活性峰。这两个峰在蛋白质印迹法中与针对PTPase保守结构域的抗（肽）血清反应，呈现出一种常见的67千道尔顿形式以及较低分子量的蛋白水解片段（31 - 56千道尔顿）。用生长抑素类似物SMS 201 - 995或BIM 23014孵育胰腺腺泡，导致膜PTPase活性受到刺激。这种刺激迅速且短暂，在添加后15分钟内达到最大水平。这两种类似物以剂量依赖方式刺激PTPase活性，对于SMS 201 - 995和BIM 23014，半最大激活分别发生在7皮摩尔和37皮摩尔，最大激活分别发生在0.1纳摩尔和0.1 - 1纳摩尔。在凝胶渗透色谱中，受刺激的膜PTPase活性也以表观分子量70千道尔顿洗脱。这两种类似物抑制[125I - Tyr3]SMS 201 - 995与胰腺腺泡细胞膜的结合，其相对效力与在刺激PTPase活性时观察到的相似。我们得出结论，胰腺腺泡细胞膜具有一种低分子量PTPase，它在涉及膜生长抑素受体激活的浓度下受到生长抑素类似物的刺激。
