Kessler F, Bennardini F, Bachs O, Serratosa J, James P, Caride A J, Gazzotti P, Penniston J T, Carafoli E
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
J Biol Chem. 1990 Sep 15;265(26):16012-9.
A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.
一种钙离子泵ATP酶已在大鼠肝细胞质膜中得到鉴定。该酶对钙离子具有高亲和力，具有P型离子泵的典型特性。与其他真核细胞质膜的钙离子泵不同，它不受钙调蛋白的刺激。在ATP存在的情况下形成的磷酸化酶的稳态浓度会因La3+而增加。该酶与针对人红细胞钙离子泵产生的单克隆抗体（mAb - 5F10）发生交叉反应。使用mAb - 5F10抗体亲和柱对该酶进行了纯化。对分离出的蛋白质进行溴化氰消化产生了两个已测序的肽段。其中一个与红细胞钙离子泵中包含的序列完全匹配，另一个与红细胞泵中的另一个结构域高度同源。尽管缺乏钙调蛋白刺激，但在变性条件下对纯化的肝脏ATP酶进行的125I - 钙调蛋白覆盖实验表明，该酶与钙调蛋白的结合甚至比红细胞泵更强。使用mAb - 5F10抗体对肝切片进行的免疫细胞化学实验表明，该酶主要位于肝细胞质膜的血窦区域。
