Sakai K, Hayashi M, Kawashima S, Akanuma H
Department of Chemistry, College of Arts and Sciences, University of Tokyo, Japan.
Biochim Biophys Acta. 1989 Oct 2;985(1):51-4. doi: 10.1016/0005-2736(89)90102-8.
The location of calcium-activated neutral proteinase (CANP) was determined in human erythrocytes by crosslinking CANP to co-localizing proteins using a photolabeling bifunctional reagent, 4,4'-dithiobisphenylazide (DTBPA). The crosslinked products were selectively isolated by immunoprecipitation with a polyclonal anti-CANP antibody and analyzed by SDS-polyacrylamide gel electrophoresis after cleavage of the crosslinkage. In the calcium-free incubation medium the main proteins crosslinked with CANP were cytosolic proteins such as hemoglobin. In the presence of calcium ions, on the other hand, membrane skeletal proteins such as spectrin, band 4.1, 4.2 and 6 proteins as well as band 3 were crosslinked with CANP. Addition of calcium ionophore further increased the amount of crosslinked membrane proteins. These results suggest that in the absence of calcium ions CANP exists diffusely in the cytoplasm and is crosslinked with cytoplasmic hemoglobin nonspecifically while in the presence of calcium ions CANP associated with membrane where it is crosslinked specifically with the lining proteins. Thus it is demonstrated biochemically that the localization of CANP is dynamic depending on the presence of calcium ions.
通过使用光标记双功能试剂4,4'-二硫代双苯叠氮（DTBPA）将钙激活中性蛋白酶（CANP）与共定位蛋白交联，确定了其在人红细胞中的位置。用多克隆抗CANP抗体通过免疫沉淀选择性分离交联产物，并在交联键裂解后通过SDS-聚丙烯酰胺凝胶电泳进行分析。在无钙孵育培养基中，与CANP交联的主要蛋白质是胞质蛋白，如血红蛋白。另一方面，在钙离子存在下，膜骨架蛋白如血影蛋白、4.1、4.2和6带蛋白以及3带蛋白与CANP交联。添加钙离子载体进一步增加了交联膜蛋白的量。这些结果表明，在没有钙离子的情况下，CANP分散存在于细胞质中，并与细胞质血红蛋白非特异性交联，而在有钙离子的情况下，CANP与膜结合，在膜上它与内膜蛋白特异性交联。因此，通过生物化学方法证明CANP的定位取决于钙离子的存在，是动态变化的。
