Poland A, Glover E
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Arch Biochem Biophys. 1988 Feb 15;261(1):103-11. doi: 10.1016/0003-9861(88)90109-9.
We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.
我们之前报道过（《生物化学杂志》（1986年）261卷，6352 - 6465页），芳烃受体的光亲和配体[¹²⁵I]-2-叠氮基-3-碘-7,8-二溴二苯并-对-二恶英，与C57BL/6小鼠的肝脏胞质溶胶部分一起孵育时，以1:1的比例标记了两条表观分子量分别为95 kDa和70 kDa且蛋白水解片段模式相似的肽段。在克隆的小鼠肝癌细胞系Hepa 1细胞的胞质溶胶部分，光亲和标记的产物几乎完全是一条95 kDa的肽段，该肽段会被一种Ca²⁺依赖性蛋白酶迅速水解为一条70 kDa的肽段以及其他片段。因此，C57BL/6小鼠肝脏和Hepa 1细胞中芳烃受体的配体结合单位是一条95 kDa的肽段，而70 kDa的片段是蛋白水解产生的假象。水解95 kDa肽段的Ca²⁺依赖性蛋白酶具有钙蛋白酶II的特性：（i）绝对需要Ca²⁺，在0.5至1.0 mM Ca²⁺时活性最高；（ii）最适pH为7.5至8.0；（iii）可被EDTA、碘乙酰胺、亮抑酶肽和L-反式-环氧琥珀酰基亮氨酰胺基（4-胍基）丁烷抑制，但不被大豆胰蛋白酶抑制剂、抑肽酶或苯甲基磺酰氟抑制。用DEAE-琼脂糖凝胶对C57BL/6小鼠的肝脏胞质溶胶进行色谱分离时，Ca²⁺依赖性蛋白酶活性（以酪蛋白或标记的95 kDa肽段为底物）在0.25 M NaCl浓度下洗脱，一种特异性蛋白酶抑制剂在0.15 M NaCl浓度下洗脱。在几种哺乳动物物种的肝脏胞质溶胶中都发现了水解95 kDa肽段的Ca²⁺依赖性蛋白酶活性。
