Tuana B S, Sunahara R K, Murphy B J, Pierce G N
Department of Pharmacology, University of Ottawa, Canada.
Biochim Biophys Acta. 1991 Jan 31;1091(2):236-41. doi: 10.1016/0167-4889(91)90067-8.
Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.
已使用⁴⁵Ca覆盖技术在离体心脏肌膜中研究了Ca²⁺结合情况。在从狗、兔、牛和猪制备的样品中，⁴⁵Ca与125 kDa和97 kDa的两种肌膜多肽结合。在DEAE离子交换柱和麦胚凝集素亲和柱上进行分级分离时，这两种Ca²⁺结合多肽与与电压门控Ca²⁺通道相关的二氢吡啶受体一起纯化。通过SDS-PAGE中的银染判断，这些多肽是分离组分中的主要蛋白质。针对纯化的狗心脏肌膜产生的抗血清表明，125 kDa和97 kDa的多肽是该膜的高度抗原性成分。该抗血清与来自兔、牛和猪的心脏肌膜制剂中的相似多肽发生交叉反应，但与肌浆网膜不发生交叉反应。针对125 kDa多肽的纯化抗体与97 kDa多肽不发生交叉反应，而针对97 kDa多肽的抗体与125 kDa多肽不发生交叉反应。125 kDa和97 kDa的多肽均结合麦胚凝集素，表明两者都是糖蛋白。这些Ca²⁺结合糖蛋白不太可能代表该膜中二氢吡啶受体-Ca²⁺通道的亚基。
