Borovsky D, Carlson D A, Griffin P R, Shabanowitz J, Hunt D F
Florida Medical Entomology Laboratory, University of Florida, Vero Beach 32962.
Insect Biochem Mol Biol. 1993 Sep;23(6):703-12. doi: 10.1016/0965-1748(93)90044-s.
Trypsin modulating oostatic factor (TMOF), a decapeptide that directly inhibits the biosynthesis of trypsin- and chymotrypsin-like enzymes in epithelial cells of mosquito midgut and indirectly inhibits vitellogenesis in anautogenous females, has been sequenced by Fourier transform mass spectrometry analysis. The peptide has a primary amino acid sequence of NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH and probably exhibits left-handed helical conformation as was shown by computer stereoview simulation. The factor is metabolized very rapidly (half-life of 1.6 h) in intact mosquitoes when injected after the blood meal. Inhibition of trypsin biosynthesis was followed in ligated abdomens, which synthesize trypsin but do not metabolise TMOF. At concentrations of 3 x 10(-9) M and 6.8 x 10(-6) M, TMOF inhibited 50 and 90% of trypsin-like enzyme biosynthesis, respectively. Several analogs of varying chain lengths were synthesized and evaluated for biological activity using dose-response curves. Switching the positions of Tyr and Asp at the N-terminus reduced the activity of the hormone, indicating that the N-terminus is important for biological activity. Removal of two to five prolines at the C-terminus also reduced activity, indicating that both the N- and C-termini are important. Synthesis of trypsin-like isozyme was followed in several insect species using [1,3-3H]diisopropyl-fluorophosphate (DFP) in the presence of tosylamide-2-phenylethyl chloromethyl ketone. Marked reduction of [1,3-3H]diisopropyl-phosphoryl-trypsin-like derivatives was noted after TMOF treatment, as assessed by polyacrylamide gel electrophoresis. These results indicate that the biosynthesis of trypsin-like enzyme in mosquitoes and other insects may be regulated by sequence-related TMOFs.
胰蛋白酶调节卵发育抑制因子（TMOF）是一种十肽，它直接抑制蚊虫中肠上皮细胞中胰蛋白酶和类胰凝乳蛋白酶的生物合成，并间接抑制非自体生殖雌蚊的卵黄生成，该因子已通过傅里叶变换质谱分析进行了测序。该肽的一级氨基酸序列为NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH，计算机立体视图模拟显示其可能呈现左手螺旋构象。当在蚊虫吸食血液后注射时，该因子在完整蚊虫体内代谢非常迅速（半衰期为1.6小时）。在结扎腹部中观察胰蛋白酶生物合成的抑制情况，结扎腹部可合成胰蛋白酶但不代谢TMOF。在浓度为3×10(-9)M和6.8×10(-6)M时，TMOF分别抑制了50%和90%的类胰蛋白酶生物合成。合成了几种不同链长的类似物，并使用剂量反应曲线评估其生物活性。在N端交换Tyr和Asp的位置会降低该激素的活性，表明N端对生物活性很重要。在C端去除两到五个脯氨酸也会降低活性，表明N端和C端都很重要。在几种昆虫物种中，在甲苯磺酰胺-2-苯乙基氯甲基酮存在的情况下，使用[1,3-3H]二异丙基氟磷酸酯（DFP）跟踪类胰蛋白酶同工酶的合成。通过聚丙烯酰胺凝胶电泳评估，TMOF处理后，[1,3-3H]二异丙基磷酰化类胰蛋白酶衍生物明显减少。这些结果表明，蚊虫和其他昆虫中类胰蛋白酶的生物合成可能受序列相关的TMOF调节。
