Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum.
Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum.
复制标题
鉴定网状质（内质网的管腔内容物）中的一组钙结合蛋白。
作者：
D. Macer;G. Koch
建立了一种分离内质网(ER)的管腔物质网状浆体的方法。从含有大量内质网的小鼠浆细胞瘤细胞系中制备了富含网状质的提取物，首先用低渗裂解提取细胞质内容物以产生富含内质网的‘壳’，然后机械裂解释放内质网内容物。提取液含有5种主要蛋白质，表观分子量分别为100、75、60、58和55(×10(3)mR)。100、75和58(X 10(3))MR分别鉴定为内纤溶酶、Bip和PD1。共聚焦荧光显微镜和亲和纯化的抗体证实了60和55(X10(3)MR蛋白的ER相关性。分离网状胞质的平衡透析法测得钙结合容量为每毫克蛋白300nmolE钙，在3 mM-Ca~(2+)时达到一半最大结合量。在钙浓度为5 mM·Ca~(2+)时，纯化的内纤溶酶每毫克蛋白结合280毫摩尔的钙。钙覆盖试验表明，除了内纤溶酶外，网状质内至少还含有三种钙结合蛋白：Bip、PDI和55×10(3)MR蛋白，其中内纤溶酶和55×10(3)MR蛋白(CRP55)占钙结合活性的主要部分。用钙离子载体处理细胞后，主要的钙结合网状纤溶酶、Bip和CRP55特异性过表达。这些研究表明，内质网的管腔内含有一系列蛋白质，能够结合毫摩尔范围内的大量钙，从而赋予内质网执行与肌肉细胞中肌浆网类似的钙存储功能的能力。
A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich 'shells' followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
肝微粒体 Ca2 依赖性 ATP 酶。
DOI：
10.1042/bj2140069
发表时间：
1983
期刊：
The Biochemical journal
影响因子：
--
作者：
Moore,PB;Kraus-Friedmann,N
通讯作者：
Kraus-Friedmann,N