Price N T, Nakielny S F, Clark S J, Proud C G
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochim Biophys Acta. 1989 Jul 7;1008(2):177-82. doi: 10.1016/0167-4781(80)90005-6.
Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the beta-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed beta H (heavy, the slower-migrating species, apparent Mr = 54,300) and beta L (light, the faster-migrating species, apparent Mr = 53,100). We confirm that two forms of eIF-2 beta are present in such preparations, but present evidence that the beta L is generated from beta H during the isolation procedure. Crude reticulocyte lysates contain only the beta H species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2 beta. The beta L species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the beta H and the beta L forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2 beta, the beta L form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the beta-subunit.
多拉基亚和瓦赫巴（《生物化学杂志》（1987年）262卷，10164 - 10170页）报道，从兔网织红细胞中制备的纯化起始因子-2（eIF-2）含有两种形式的β亚基。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）判断，这些形式的表观分子量不同，因此分别称为βH（重链，迁移较慢的种类，表观Mr = 54,300）和βL（轻链，迁移较快的种类，表观Mr = 53,100）。我们证实此类制剂中存在两种形式的eIF-2β，但现有证据表明βL是在分离过程中由βH产生的。从使用抗eIF-2β抗血清对SDS-PAGE分离的网织红细胞蛋白进行免疫印迹判断，粗制网织红细胞裂解物仅含有βH种类。βL种类在纯化过程早期使用的硫酸铵分级分离步骤之后出现，但如果在纯化过程中使用的缓冲液中包含蛋白酶抑制剂混合物，则不明显，这表明它是分离过程中产生的蛋白水解降解产物。克利夫兰图谱未能揭示这两种种类之间的任何差异。βH和βL形式都被酪蛋白激酶-2磷酸化，并且通过一维和二维肽图谱判断，每种种类在相同位点磷酸化。由于酪蛋白激酶-2使eIF-2β中的丝氨酸-2磷酸化，βL形式必定仍包含N端区域，推测是由β亚基羧基末端的有限蛋白水解产生的。
