Fiertek D, Gromowska M, Andersen A S, Hansen P H, Majewski T, Izdebski J
Department of Chemistry, University of Warsaw, Poland.
J Pept Sci. 2000 Aug;6(8):413-9. doi: 10.1002/1099-1387(200008)6:8<413::AID-PSC268>3.0.CO;2-Z.
Insulin and C-peptide were extracted and purified from fox (Vulpes vulpes) pancreas using gel filtration, ion-exchange chromatography and HPLC. Chromatographic data for the insulin, as well as for its oxidized and carboxymethylated chains proved it to be identical to that of polar fox (Alopex lagopus) and dog. The sequence analysis of a peptide which was assumed to be the corresponding C-peptide revealed that it comprises 23 amino acid residues and is identical to the C-peptide fragment isolated from dog pancreas: it differs from polar fox C-peptide by a single substitution (Asp-->Glu). mRNA was isolated from pancreatic tissue and cDNA was obtained by reverse transcription. A polymerase chain reaction was performed using gene-specific primers to obtain a DNA fragment corresponding to part of fox proinsulin. DNA sequencing revealed 100% identity to dog proinsulin at the protein level, although some amino acids were encoded by different codons. The total sequence of proinsulin was deduced from these results.
使用凝胶过滤、离子交换色谱法和高效液相色谱法从狐狸（赤狐）胰腺中提取并纯化胰岛素和C肽。胰岛素及其氧化链和羧甲基化链的色谱数据证明其与北极狐和狗的胰岛素相同。对一个被认为是相应C肽的肽段进行序列分析，结果显示它由23个氨基酸残基组成，与从狗胰腺中分离出的C肽片段相同：它与北极狐C肽的区别仅在于一个氨基酸的替换（天冬氨酸→谷氨酸）。从胰腺组织中分离出mRNA，并通过逆转录获得cDNA。使用基因特异性引物进行聚合酶链反应，以获得对应于狐狸胰岛素原部分的DNA片段。DNA测序显示，尽管某些氨基酸由不同密码子编码，但在蛋白质水平上与狗胰岛素原具有100%的同一性。根据这些结果推导了胰岛素原的完整序列。
