Talarico T L, Barkocy-Gallagher G A, Ray P H, Bassford P J
Department of Molecular Genetics and Microbiology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Biochem Biophys Res Commun. 1993 Dec 30;197(3):1154-66. doi: 10.1006/bbrc.1993.2598.
Processing of 37 precursor maltose-binding protein (preMBP) species by purified signal peptidase I (SPase I) was assayed. The in vitro reaction was inefficient compared to processing in Escherichia coli cells. The extent of preMBP processing in vitro was higher when SPase I was present during translation as compared to processing after translation was arrested by chloramphenicol. Complete conversion of wild-type (wt) preMBP (greater than 90%) to mature protein required 4300-fold more enzyme than substrate during a 15 min reaction. Most preMBP species with alterations in the signal peptide processing region that were efficiently processed (greater than 85%) in vivo were also processed in vitro, although the efficiency of processing was usually lower than the corresponding in vivo value. Increasing the level of SPase I in the in vitro reaction often increased the extent of preMBP processing. A number of amino acid substitutions in the processing region that drastically reduced or eliminated processing in vivo also eliminated processing in vitro. Processing occurred at an alternate site in some mutant preMBP species in vivo, but this event occurred very inefficiently in vitro. Amino acid substitutions in the hydrophobic core or in the charged regions at the N-terminus of the signal peptide and early mature region of preMBP slightly reduced in vitro processing as compared to processing of wt preMBP, regardless of their effect on secretion in vivo.
对37种前体麦芽糖结合蛋白（preMBP）进行了纯化信号肽酶I（SPase I）处理的检测。与在大肠杆菌细胞中的处理相比，体外反应效率较低。与氯霉素阻止翻译后进行处理相比，翻译过程中存在SPase I时，preMBP的体外处理程度更高。在15分钟的反应中，野生型（wt）preMBP完全转化为成熟蛋白（大于90%）所需的酶量比底物多4300倍。大多数在体内能有效处理（大于85%）的信号肽加工区域发生改变的preMBP物种在体外也能被处理，尽管处理效率通常低于相应的体内值。在体外反应中增加SPase I的水平通常会提高preMBP的处理程度。一些在加工区域的氨基酸替换在体内会大幅降低或消除处理，在体外也会消除处理。在体内，一些突变preMBP物种在一个替代位点发生处理，但在体外这种情况发生的效率非常低。与wt preMBP的处理相比，preMBP信号肽疏水核心或N端带电区域以及早期成熟区域的氨基酸替换在体外会略微降低处理效率，无论它们对体内分泌的影响如何。
