Takase K, Mizuno H, Yamane K
Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki, Japan.
J Biol Chem. 1988 Aug 15;263(23):11548-53.
Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.
已知枯草芽孢杆菌的成熟α-淀粉酶是由其前体通过去除NH2末端41个氨基酸序列形成的。为了研究这种加工机制，对在枯草芽孢杆菌中克隆并表达的枯草芽孢杆菌N7α-淀粉酶的细胞外形式进行了分析。从L肉汤对数期培养物中分离出的主要形式（形式N34）的NH2末端对应于起始甲硫氨酸的第34位，但通过天然凝胶电泳判断似乎存在微异质性。稳定期培养物中的主要形式在第40位（形式N40）或第42位（形式N42）具有NH2末端，并且是均一的。较大形式向较小形式的转化可以在培养上清液或部分纯化的样品中实现。过程N34→N40被EDTA抑制；N40→N42被Ca2+促进。苯甲基磺酰氟抑制前者而不抑制后者过程。这些结果表明信号肽酶切割位点30至35是-Ala-Ala-Ala-Ser-Ala-Glu-Thr-（箭头或更上游），并且蛋白水解成熟发生在分泌后，这涉及至少两种不同的加工酶。
