STUDIES ON ANIMAL alpha-AMYLASE INHIBITOR PRODUCED BY STREPTOMYCES

由亚芽链霉菌T-76产生的蛋白性α -酰化酶抑制剂（T-76- i）被TPCK胰蛋白酶羧甲基化消化。所得片段在反相色谱柱上进行高效液相色谱分离，并分别应用于气相氨基酸测序仪。测定了成熟T-76-I的54个氨基酸残基（约70%）。利用人工合成的寡核苷酸探针将T-76-I基因克隆到一个1.2kb的Sau 3AI片段中，该片段在密码子第3个字母的位置含有肌苷，对应于抑制剂的n端。插入片段的核苷酸序列数据表明，该片段包含330个核苷酸的单独开放阅读框，编码110个氨基酸的多肽，Mr为11,306道尔顿。由核苷酸序列推断出的氨基酸序列表明，假定的T-76-I前蛋白将信号肽的33个氨基酸延伸到由77个氨基酸组成的成熟T-76-I蛋白的n -端，Mr为8,082道尔顿。预t -76- i蛋白被证明在分泌过程中被加工。

Proteinaceous alpha-anylase inhibitor(T-76-I) produced by Streptomyces nitrosporeus T-76 was corboxymethylated and digested by TPCK trypsin. The resulting fragments were separated using HPLC on reverse-phase colum and each of then was applied to gas phase amino acid sequencer. Fifty-four amino acid residues(approx, 70%) of mature T-76-I were determined.T-76-I gene was cloned in a 1.2kb Sau 3AI fragment using a synthetic oligonucleotide probe, which contained inosine at the position of 3rd letter of codons, corresponding to the N-teminal end of the inhibitor. The nucleotide sequence data of the inserted fragment showed that the fragment contained sole open reading frame of 330 nucleotide encoding a polypeptide of 110 amino acids with a Mr of 11,306 dalton. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-T-76-I protein extends 33 amino acids of signal peptide to the N-teminus of the mature T-76-I protein consisting of 77 amino acids with a Mr of 8,082 dalton. The pre-T-76-I protein is demonstrated to be processed during secrerion.