Noe B D, Fletcher D J, Bauer G E, Weir G C, Patel Y
Endocrinology. 1978 Jun;102(6):1675-85. doi: 10.1210/endo-102-6-1675.
No information is at present available on the mode of SRIF biosynthesis. Since anglerfish pancreatic islet tissue is comprised of approximately 30% D cells, we have examined this tissue for SRIF synthesis . The following known differences in amino acid composition of islet peptides were used advantageously in this study: anglerfish proinsulin: Trp-0, Ile-2, Cys-6; anglerfish glucagon: Trp-1, Ile-0, Cys-0; mammalian SRIF: Trp-1, Ile-0, Cys-2. After incubating islet tissue with [3H]tryptophan and [14C]isoleucine or [35S]cystine for various time periods, proteins were extracted in 2 M acetic acid and desalted by Bio-Gel P-2 gel filtration. P-2 void volume proteins were then subjected to P-10 gel filtration and isolated by polyacrylamide gel electrophoresis (PAGE) at alkaline pH. The predominant amount of the immumoreactive SRIF in the extracts appeared in a peak eluting just before the salt volume on P-10 filtration and migrated slowly toward the cathode during PAGE. The behavior of synthetic SRIF was identical. The anglerfish SRIF immunoreactive peptide could be labeled with Trp and Cys but not Ile during incubations longer than 1 h. The Trp- and Cys-labeled peptide could be bound on columns to which the immunoglobulin fraction of antisera to SRIF had been complexed. Cycloheximide inhibited isotope incorporation into all islet proteins. These results indicate that islet SRIF is synthesized in situ. Moreover, the immunological activity, size, and charge characteristics of anglerfish islet SRIF appear to be similar to those of mammalian hypothalamic SRIF. When islets were subjected to short pulse incubations with labeled Trp and Cys, only peptides eluting in the 7,000-13,000 dalton portion of the filtration eluate became labeled. No appreciable isotope incorporation into SRIF was observed. However, when pulse incubations were followed by incubation in the presence of cycloheximide or excess unlabeled amino acids in isotope-free medium (chase), the incorporation of Trp and Cys into SRIF increased with the length of chase, suggesting the participation of a larger precursor in SRIF synthesis.
目前尚无关于生长抑素生物合成模式的信息。由于安康鱼胰岛组织约由30%的D细胞组成,我们已对该组织进行生长抑素合成的检测。在本研究中,有利地利用了以下已知的胰岛肽氨基酸组成差异:安康鱼胰岛素原:色氨酸-0、异亮氨酸-2、半胱氨酸-6;安康鱼胰高血糖素:色氨酸-1、异亮氨酸-0、半胱氨酸-0;哺乳动物生长抑素:色氨酸-1、异亮氨酸-0、半胱氨酸-2。用[³H]色氨酸、[¹⁴C]异亮氨酸或[³⁵S]胱氨酸孵育胰岛组织不同时间段后,蛋白质在2M乙酸中提取,并用Bio-Gel P-2凝胶过滤进行脱盐。然后将P-2空体积蛋白进行P-10凝胶过滤,并在碱性pH下通过聚丙烯酰胺凝胶电泳(PAGE)分离。提取物中主要的免疫反应性生长抑素出现在P-10过滤时刚好在盐峰之前洗脱的一个峰中,并且在PAGE过程中缓慢向阴极迁移。合成生长抑素的行为相同。在孵育超过1小时的过程中,安康鱼生长抑素免疫反应性肽可以用色氨酸和半胱氨酸标记,但不能用异亮氨酸标记。色氨酸和半胱氨酸标记的肽可以结合到与抗生长抑素血清的免疫球蛋白部分复合的柱上。放线菌酮抑制所有胰岛蛋白的同位素掺入。这些结果表明胰岛生长抑素是原位合成的。此外,安康鱼胰岛生长抑素的免疫活性、大小和电荷特性似乎与哺乳动物下丘脑生长抑素相似。当胰岛用标记的色氨酸和半胱氨酸进行短脉冲孵育时,只有在过滤洗脱液7000 - 13000道尔顿部分洗脱的肽被标记。未观察到生长抑素中有明显的同位素掺入。然而,当脉冲孵育后在放线菌酮存在下或在无同位素培养基中用过量未标记氨基酸进行孵育(追踪)时,色氨酸和半胱氨酸掺入生长抑素的量随追踪时间的延长而增加,表明有一个更大的前体参与生长抑素的合成。