Hayashi H, Owada M K, Sonobe S, Kakunaga T
Department of Oncogene Research, Osaka University, Japan.
J Biol Chem. 1989 Oct 15;264(29):17222-30.
Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.
通过用5 mM乙二醇双乙醚二胺四乙酸（EGTA）处理，从人胎盘中溶解出两种不同的68 kDa蛋白质，分别命名为68K-I（等电点6.4）和68K-II（等电点5.6）。在pH 7.4条件下进行DE52纤维素柱层析时，EGTA洗脱液中的68K-I在未吸附级分中回收，而68K-II保留在柱上并用0.2 M氯化钠洗脱。通过进一步的羟基磷灰石和阳离子交换层析，68K-I蛋白的纯度达到95%以上，而68K-II蛋白则通过凝胶过滤和羟基磷灰石层析进一步纯化。部分氨基酸序列数据表明，68K-I蛋白是一种与脂皮质素I具有相同序列的新型蛋白，68K-II与人p68/67-kDa钙电蛋白相同（Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21 - 27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664 - 668）。在微摩尔浓度的Ca2+存在下，这两种蛋白都能与酸性磷脂、磷脂酰丝氨酸和/或磷脂酰肌醇结合，但不与磷脂酰胆碱结合。68K-I优先与磷脂酰肌醇结合而非磷脂酰丝氨酸，而68K-II仅与磷脂酰丝氨酸结合。68K-I和68K-II都能抑制磷脂酶A2的活性，且只有在100 mM氯化钾存在时才能检测到68K-II的抑制作用。发现68K-I能以Ca2+依赖（1 mM）的方式与F-肌动蛋白结合，而68K-II则不能。此外，68K-I能在体外被fps激酶和表皮生长因子受体/激酶在酪氨酸残基上磷酸化，后一反应依赖于Ca2+和表皮生长因子。用亲和纯化的抗68K-I和抗68K-II抗体进行的蛋白质印迹分析表明，68K-I仅存在于某些组织中，尤其是人胎盘，而68K-II存在于许多人和大鼠组织中。
