Sarkadi B, Enyedi A, Gárdos G
Biochim Biophys Acta. 1987 May 12;899(1):129-33. doi: 10.1016/0005-2736(87)90247-1.
In inside-out red cell membrane vesicles trypsin digestion reduces the molecular mass of the 32P-labeled acyl-phosphate intermediate of the calcium pump from the original 140 kDa to about 80 kDa with a simultaneous activation of the calcium uptake. This process is slightly stimulated by the presence of calcium, as compared to EGTA, or EGTA + vanadate, but the proteolytic pattern is similar under all these conditions. However, trypsin degradation of the 80 kDa polypeptide, resulting in the loss of calcium transport activity and 32P-phosphoenzyme formation, is rapid in the presence of calcium, inhibited by EGTA and almost fully blocked by EGTA + vanadate. In the presence of these latter ligands, probably locking the calcium pump in an E2 conformation, the 80 kDa protein becomes insensitive even to excessive digestion by the non-specific protease, pronase. The data indicate major changes in the molecular arrangement of the calcium pump protein when transformed from a calcium-liganded (E1) to an E2 conformation.
在内外翻转的红细胞膜囊泡中，胰蛋白酶消化可使钙泵的32P标记的酰基磷酸中间体的分子量从原来的140 kDa降至约80 kDa，同时激活钙摄取。与EGTA或EGTA +钒酸盐相比，钙的存在会轻微刺激这一过程，但在所有这些条件下蛋白水解模式相似。然而，80 kDa多肽的胰蛋白酶降解会导致钙转运活性丧失和32P -磷酸酶形成，在有钙的情况下降解迅速，被EGTA抑制，几乎完全被EGTA +钒酸盐阻断。在存在后一种配体的情况下，可能会将钙泵锁定在E2构象，80 kDa的蛋白质甚至对非特异性蛋白酶链霉蛋白酶的过度消化也变得不敏感。数据表明，当钙泵蛋白从钙结合（E1）构象转变为E2构象时，其分子排列发生了重大变化。
