Karamyshev A L, Kalinin A E, Khmel'nitskiĭ M I, Shliapnikov M G, Ksenzenko V N, Nesmeianova M A
Mol Biol (Mosk). 1994 Mar-Apr;28(2):374-82.
The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.
研究了N端氨基酸取代对大肠杆菌碱性磷酸酶生物合成的影响。通过在phoA基因内的相应位置产生琥珀突变，并在产生琥珀抑制tRNA的大肠杆菌菌株中表达该突变基因，获得了用Ser、Gln、Tyr、Leu、Gly、Ala、Glu、Phe、His、Cys、Lys和Pro取代Arg(+1)的结果。所有突变蛋白均显示能跨细胞质膜转运并具有酶活性。在+1位引入Pro会干扰信号肽的切割，而与野生型蛋白相比，插入其他氨基酸不会改变加工速率。所有氨基酸取代都会影响碱性磷酸酶同工酶的组成。还获得了一些关于蛋白酶特异性的实验证据，该蛋白酶在碱性磷酸酶成熟过程中切割掉N端的Arg。
