SITE OF REACTION ON RIBOSOMAL-PROTEIN L27 WITH AN AFFINITY LABEL DERIVATIVE OF TRANSFER-RNA-F(MET)
SITE OF REACTION ON RIBOSOMAL-PROTEIN L27 WITH AN AFFINITY LABEL DERIVATIVE OF TRANSFER-RNA-F(MET)
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DOI：
10.1016/0014-5793(76)80112-3
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发表时间：
1976-01-01
期刊：
影响因子：
3.5
通讯作者：
CZERNILOFSKY, AP
中科院分区：
文献类型：
--
作者：
COLLATZ, E;KUCHLER, E;CZERNILOFSKY, AP
PNPC-Met-tRNAmf et** 最近被用于从E. eoli，主要标记的蛋白是L27和L15 [1，2]。标记反应的特异性通过其对天然mRNA [1]和起始因子[2]的依赖性以及嘌呤霉素的抑制作用[3]得到证明。为了进一步研究反应的位点，通过指纹技术对来自标记蛋白L27的胰蛋白酶肽进行分析。仅在两个斑点中检测到放射性。通过依次用胰蛋白酶-嗜热菌蛋白酶-链霉蛋白酶或胰蛋白酶-胰凝乳蛋白酶-链霉蛋白酶消化标记的蛋白质获得完全的酶促水解，产生独特的终产物，其通过其相对Rf值与合成的Lys-NH-CO-NH-Met化合物相比较。由此得出结论，亲和标记反应发生在蛋白质L27的一个或两个不同的位点与赖氨酸的e-氨基。
PNPC-Met-tRNAmf et** was recently used for affinity labelling of ribosomal proteins from the P-site in E. eoli and the predominantly labelled proteins were L27 and L15 [1, 2]. The specificity of the labelling reaction was proved by its dependence on natural mRNA [1] and initiation factors [2] and by the inhibition with puromycin [3]. To further investigate the site of the reaction, analyses of the tryptic peptides from labelled protein L27 were performed by a fingerprinting technique. Radioactivity was detected in only two spots. Complete enzymatic hydrolysis was obtained by digesting the labelled protein sequentially with either trypsin-thermolysine-pronase or trypsin-chymotrypsin-pronase yielding a unique endproduct which, by its relative Rf-values, compares to a synthesized Lys-NH-CO-NH-Met compound. It was concluded that the affinity labelling reaction occurs at one or two distinct sites of protein L27 with the e-amino groups of lysine.