Chou J Z, Chait B T, Wang R, Kreek M J
Laboratory on the Biology of Addictive Diseases, Rockefeller University, New York, NY 10021, USA.
Peptides. 1996;17(6):983-90. doi: 10.1016/0196-9781(96)00154-4.
The biotransformation in human blood in vitro of three dynorphin A (Dyn A) peptides was studied by matrix assisted laser desorption mass spectrometry to determine whether the natural peptide, Dyn A(1-17), is biotransformed differently from Dyn A (1-13), the natural sequence shortened form used in numerous neurobiological and pharmacological studies. In addition to studies of Dyn A(1-17), a natural product from prodynorphin and Dyn A(1-13), a natural sequence truncation of Dyn A(1-17), Dyn A(1-10)amide, a synthetic analogue of Dyn A(1-17) presumed to be protected from rapid biotransformation was also studied Synthetic Dyn A peptides were incubated in freshly drawn blood for various periods of time prior to mass spectrometric analysis. Several peptide products were identified from each precursor; the time profiles of appearance and disappearance of the major products were followed. Substantial differences in products and especially in the rate of biotransformation were observed between the processing of Dyn A(1-17) and the two shorter Dyn A peptides, Dyn A(1-13) and Dyn A(1-10)amide. Significant amounts of the natural Dyn A(1-17) survived 4 h of incubation (half-life 3 h). Dyn A (2-17), a major processed product of Dyn A(1-17) in blood, continued to accumulate during the 4-h incubation period. By contrast, both Dyn A(1-13) and Dyn A(1-10) amide were biotransformed very rapidly with half-lives of < 1 min and 10 min, respectively. Most of the products from these two peptide precursors were also further processed rapidly, with the exception of Dyn A(4-12) and Dyn A(4-10)amide, which were detected for over 2 h. Dyn A(1-6) was found as a minor biotransformation product from all three precursor peptides. These findings suggest that an important function of the four C-terminal amino acid residues of the natural form, Dyn A(1-17) [compared to Dyn A(1-13)], is to stabilize or protect the peptide from biotransformation by enzymes, by preserving a natural hairpin structure possibly near the carboxyl-terminus.
通过基质辅助激光解吸质谱法研究了三种强啡肽A（Dyn A）肽在人血液中的体外生物转化，以确定天然肽Dyn A（1-17）与Dyn A（1-13）（在众多神经生物学和药理学研究中使用的天然序列缩短形式）的生物转化是否不同。除了对来自前强啡肽的天然产物Dyn A（1-17）和Dyn A（1-13）（Dyn A（1-17）的天然序列截短形式）进行研究外，还研究了Dyn A（1-10）酰胺，一种推测可免受快速生物转化的Dyn A（1-17）合成类似物。在进行质谱分析之前，将合成的Dyn A肽在新鲜采集的血液中孵育不同时间。从每个前体中鉴定出几种肽产物；跟踪主要产物出现和消失的时间曲线。在Dyn A（1-17）与两种较短的Dyn A肽Dyn A（1-13）和Dyn A（1-10）酰胺的处理过程中，观察到产物尤其是生物转化速率存在显著差异。大量天然Dyn A（1-17）在孵育4小时后仍存在（半衰期3小时）。Dyn A（2-17）是血液中Dyn A（1-17）的主要加工产物，在4小时孵育期内持续积累。相比之下，Dyn A（1-13）和Dyn A（1-10）酰胺均迅速发生生物转化，半衰期分别<1分钟和10分钟。这两种肽前体的大多数产物也进一步快速加工，除了Dyn A（4-12）和Dyn A（4-10）酰胺，它们被检测到超过2小时。Dyn A（1-6）被发现是所有三种前体肽的次要生物转化产物。这些发现表明，天然形式Dyn A（1-17）[与Dyn A（1-13）相比]的四个C末端氨基酸残基的一个重要功能是通过保留可能靠近羧基末端的天然发夹结构来稳定或保护肽免受酶的生物转化。
