King L E, Gates R E
Arch Biochem Biophys. 1985 Oct;242(1):146-56. doi: 10.1016/0003-9861(85)90488-6.
Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.
需要钙离子的蛋白酶可降解胞质蛋白和整合膜蛋白，还能通过有限的蛋白水解作用改变某些蛋白激酶的活性。细胞裂解时，一种需要钙离子的蛋白酶会将表皮生长因子（EGF）受体（一种具有内在激酶活性的整合膜蛋白）从其170 kDa的形式降解为150 kDa的形式。这种需要钙离子的蛋白酶具有钙激活中性蛋白酶（CANP）的所有特征。为了证明CANP是体外唯一负责降解天然EGF受体的蛋白酶，从牛肺中高度纯化了CANP。这种亲和纯化的CANP具有先前针对其他CANP所描述的特性：80 kDa和30 kDa的异二聚体；最适中性pH；毫摩尔浓度的钙离子激活；以及被内源性热稳定蛋白质抑制剂、亮抑酶肽和巯基烷基化剂抑制。使用通过与125I-EGF共价连接而标记的EGF受体，这种纯化的CANP在A-431细胞膜中从天然受体定量生成150 kDa的形式。与天然受体一样，由内源性需要钙离子的蛋白酶、CANP、胰凝乳蛋白酶和弹性蛋白酶产生的150 kDa受体形式都能够进行EGF刺激的自磷酸化。当通过三种外源添加的蛋白酶产生150 kDa受体形式时，用[γ-32P]ATP进行自磷酸化然后用胰蛋白酶处理产生的32P标记肽并不相同。然而，由CANP或内源性需要钙离子的蛋白酶产生的自磷酸化150 kDa受体形式的胰蛋白酶32P标记肽是相同的。这些数据表明，CANP与细胞裂解时负责从天然EGF受体产生自磷酸化150 kDa受体形式的内源性需要钙离子的蛋白酶相同。
