Kovács T, Corvazier E, Papp B, Magnier C, Bredoux R, Enyedi A, Sarkadi B, Enouf J
U-348 Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France.
J Biol Chem. 1994 Feb 25;269(8):6177-84.
Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.
先前已在血小板中鉴定出两种肌浆网/内质网Ca(2+)-ATP酶（SERCAs）：100 kDa的SERCA2b和97 kDa的SERCA3亚型。对酰基磷酸中间体（E-P）形成以及血小板Ca(2+)-ATP酶及其在可控胰蛋白酶消化后的蛋白水解片段的免疫反应性进行分析，结果显示存在另一种97 kDa的Ca(2+)-ATP酶，它在SDS-聚丙烯酰胺凝胶上与SERCA3迁移率相同。在4℃下胰蛋白酶与膜蛋白的比例为0.025时，使用已证明可识别97 kDa Ca(2+)-ATP酶的PL/IM 430抗Ca(2+)-ATP酶抗体，可检测到SERCA家族中先前未知的73 kDa、68 kDa和40 kDa的胰蛋白酶片段。73 kDa和68 kDa的片段是40 kDa片段的前体。73 kDa片段的Ca(2+)-依赖性磷酸标记以及另一种针对SERCA1产生的抗体对所有这些蛋白水解产物的免疫染色确定了97 kDa亲本酶的SERCA性质。与SERCA3相关的形成E-P的80 kDa胰蛋白酶片段在胰蛋白酶消化过程中出现的时间进程与73 kDa、68 kDa和40 kDa的片段不同。在37℃下胰蛋白酶与膜蛋白的比例为0.125时，它在消化5分钟时达到最大水平，而73 kDa、68 kDa和40 kDa的片段在胰蛋白酶处理2分钟时完全降解。这种80 kDa的蛋白既未被PL/IM 430免疫染色，也未被抗SERCA1抗体免疫染色。在一些巨核母细胞样和淋巴母细胞样细胞系中也发现了类似结果。所有这些数据表明存在两种不同的胰蛋白酶裂解模式，归因于两种97 kDa的SERCA亚型，并指出在不同的人类非肌肉细胞中存在多SERCA系统。
