Milner R E, Baksh S, Shemanko C, Carpenter M R, Smillie L, Vance J E, Opas M, Michalak M
Cardiovascular Disease Research Group, University of Alberta, Edmonton, Canada.
J Biol Chem. 1991 Apr 15;266(11):7155-65.
The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.
研究了肌集钙蛋白和钙网蛋白在平滑肌和非肌肉组织中的分布。用抗钙网蛋白和抗肌集钙蛋白抗体探测内质网蛋白的免疫印迹显示，大鼠肝脏内质网中仅存在钙网蛋白。从富含肌浆网的子宫平滑肌中分离出的膜组分含有一条与抗钙网蛋白抗体发生免疫反应但与抗肌集钙蛋白抗体不发生免疫反应的蛋白带。通过45Ca2+覆盖和“全染”技术进一步证实了这些膜组分中存在钙网蛋白。通过用抗钙网蛋白抗体对平滑肌细胞进行间接免疫荧光染色，钙网蛋白也定位于平滑肌肌浆网。此外，发现肝脏和子宫平滑肌都含有高水平的编码钙网蛋白的mRNA，而未检测到编码肌集钙蛋白的mRNA。我们采用硫酸铵沉淀法，随后进行Mono Q快速蛋白质液相色谱法，以此从全组织匀浆中分离肌集钙蛋白和钙网蛋白，即使它们存在于同一组织中，也能将它们清晰地彼此分离。从兔和牛肝脏、兔脑、兔和猪子宫以及牛胰腺中分离出钙网蛋白，并通过其氨基末端氨基酸序列进行鉴定。在全肝组织的制剂中检测不到肌集钙蛋白；在子宫平滑肌的硫酸铵提取物中仅能检测到极少量的肌集钙蛋白。我们得出结论，钙网蛋白而非肌集钙蛋白是肝脏内质网和子宫平滑肌肌浆网中的主要Ca2+结合蛋白。肌集钙蛋白和钙网蛋白可能在肌浆网和内质网腔中发挥平行功能。
