Gilchrist J S, Pierce G N
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
J Biol Chem. 1993 Feb 25;268(6):4291-9.
Recent evidence suggests that nuclei possess Ca2+ transport mechanisms to regulate nucleoplasmic/cytosolic Ca2+ gradients. We, therefore, investigated the possibility that Ca(2+)-binding proteins may also exist within the nucleus. Electrophoretic analysis revealed the presence of an acidic 93-kDa protein (p93) in the membranes of isolated nuclei. p93 stained blue with "Stains-All" in SDS-polyacrylamide gels and was the major 45Ca(2+)- and ruthenium red-binding nuclear envelope protein in electroblot overlays. p93 was resistant to extraction by 6 M urea but was solubilized in 2% Triton X-100. Citric acid was highly effective in removing the outer nuclear membrane (ER) with concomitant reduction (< 10-fold) of mannose-6-phosphatase activity, but not p93. 45Ca(2+)-binding assays of purified p93 revealed the presence of high capacity Ca(2+)-binding sites comparable to calreticulin. This evidence strongly suggests that p93 is a major Ca(2+)-binding protein of the inner nuclear envelope membrane. Partial amino acid sequence analysis revealed that p93 was close to 100% homologous with a recently identified ER Ca(2+)-binding protein known as calnexin. It is likely, therefore, that p93 is calnexin. However, mild CHAPS detergent treatment of nuclear envelopes and ER revealed distinctly different solubility properties of each membrane for the extraction of p93. This, together with the citrate data, strongly suggests that p93/calnexin, in isolated nuclear envelopes, is mostly bound to the inner membrane. It is possible that p93 may be involved with the regulation of Ca2+ transients between the nucleoplasm and perinuclear space.
最近的证据表明，细胞核拥有钙转运机制来调节核质/胞质钙梯度。因此，我们研究了细胞核内是否也存在钙结合蛋白的可能性。电泳分析显示，在分离的细胞核膜中存在一种酸性93 kDa蛋白（p93）。在SDS-聚丙烯酰胺凝胶中，p93用“全染剂”染成蓝色，并且是电印迹覆盖物中主要的45Ca(2+)和钌红结合核膜蛋白。p93对6 M尿素的提取具有抗性，但可溶于2% Triton X-100。柠檬酸在去除核外膜（内质网）方面非常有效，同时伴随甘露糖-6-磷酸酶活性降低（< 10倍），但对p93无效。对纯化的p93进行的45Ca(2+)结合测定显示存在与钙网蛋白相当的高容量钙结合位点。这一证据强烈表明p93是内核膜的主要钙结合蛋白。部分氨基酸序列分析表明，p93与最近鉴定的一种称为钙连蛋白的内质网钙结合蛋白几乎100%同源。因此，p93很可能就是钙连蛋白。然而，用温和的CHAPS去污剂处理核膜和内质网，发现每种膜对p93提取的溶解性明显不同。这与柠檬酸盐数据一起，强烈表明在分离的核膜中，p93/钙连蛋白主要与内膜结合。p93可能参与调节核质和核周空间之间的钙瞬变。
