Webb R, Dormer R L
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Biochim Biophys Acta. 1995 Jan 26;1233(1):1-6. doi: 10.1016/0005-2736(94)00215-b.
Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca2+,Mg-ATPase activity only after UV irradiation and the inhibition was prevented by inclusion of 5 mM ATP in the labelling reaction. Rough ER proteins of apparent molecular masses 141, 111, 100, 84, 69, 55 and 47 kDa were detected following photoaffinity-labelling with 8-azido-[alpha-32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca2+,Mg-ATPase isoforms previously demonstrated immunologically [1]. Immunoprecipitation of rough ER proteins by a SERCA-2b-specific antibody showed that the two ATPase bands were photoaffinity-labelled. Photoaffinity labelling of the 111 and 100 kDa proteins was: (a) abolished when Ca2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.
利用8-叠氮基-ATP进行特异性光亲和标记，鉴定出胰腺粗面内质网ATP结合蛋白，包括SERCA-2b Ca2 +,Mg-ATPase的两种同工型。8-叠氮基-ATP仅在紫外线照射后不可逆地抑制Ca2 +,Mg-ATPase活性，并且在标记反应中加入5 mM ATP可防止这种抑制。用8-叠氮基-[α-32P]ATP进行光亲和标记后，检测到表观分子量为141、111、100、84、69、55和47 kDa的粗面内质网蛋白。111 kDa和100 kDa处的两条带在分子量上与先前通过免疫方法证实的两种SERCA-2b Ca2 +,Mg-ATPase同工型相对应。用SERCA-2b特异性抗体对粗面内质网蛋白进行免疫沉淀表明，这两条ATPase带被光亲和标记。111和100 kDa蛋白的光亲和标记情况如下：(a) 用EDTA处理粗面内质网膜使Ca2 +,Mg-ATPase活性失活时，标记被消除；(b) 被Ca2 +,Mg-ATPase抑制剂钒酸盐抑制；(c) 不受毒胡萝卜素影响。数据表明，胰腺粗面内质网含有两种SERCA-2b Ca2 +,Mg-ATPase同工型，其ATP结合特性易受钒酸盐抑制，但不受毒胡萝卜素抑制。
