Goren A, Zohar Y, Fridkin M, Elhanati E, Koch Y
National Center for Mariculture, Israel Oceanographic and Limnological Research Institute, Eilat.
Gen Comp Endocrinol. 1990 Aug;79(2):291-305. doi: 10.1016/0016-6480(90)90115-3.
The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH) and mammalian leuteinizing hormone-releasing hormone (LHRH) by pituitary bound enzymes were studied in the gilthead seabream, Sparus aurata. sGnRH and LHRH were incubated for different periods of time with membrane or cytosolic fractions of pituitary homogenates. At the end of the incubation, the degradation mixture was fractionated on reverse-phase high-pressure liquid chromatography. The degradation products were identified by comparing their retention times to those of synthesized GnRH fragments and by analyzing their amino acid composition. The main GnRH degradative activity resides in the cytosolic fraction of the pituitary homogenate. Both sGnRH and LHRH are rapidly degraded by pituitary cytosol, with 78.3 and 87.7% of the peptides, respectively, cleaved after 3 hr of incubation. Maximal degradation of sGnRH occurred at a pH range of 7 to 8. The main initial products of degradation of sGnRH and LHRH are the 1-5, 6-10, and 1-9 fragments. This suggests the involvement of two site-specific peptidases, a Tyr5-Gly6 endopeptidase and a Pro9-Gly10NH2 peptidase or postproline cleaving enzyme. While the 1-6 and 1-9 fragments undergo rapid secondary degradation, the 1-5 is relatively stable. Competition experiments suggest that the endopeptidase cleaving the sGnRH at the Tyr5-Gly6 bond is not specific to the neuropeptide and is probably a general proteolitic enzyme. However, the cleavage at the 9-10 bond has a high degree of specificity to the Pro9-Gly10NH2 sequence found in sGnRH. The two proposed pituitary peptidases of S. aurata have some characteristics similar to those of rat hypophyseal and hypothalamic GnRH cleaving enzymes. No differences are found in hypophyseal GnRH degradative activity between females with occytes undergoing previtellogenesis or advanced stages of vitellogenesis.
在金头鲷（Sparus aurata）中研究了垂体结合酶对天然鲑鱼促性腺激素释放激素（sGnRH）和哺乳动物促黄体生成素释放激素（LHRH）的降解模式和动力学。将sGnRH和LHRH与垂体匀浆的膜或胞质部分孵育不同时间。孵育结束时，将降解混合物在反相高压液相色谱上进行分离。通过将其保留时间与合成的GnRH片段的保留时间进行比较并分析其氨基酸组成来鉴定降解产物。主要的GnRH降解活性存在于垂体匀浆的胞质部分。sGnRH和LHRH都被垂体胞质溶胶迅速降解，孵育3小时后，分别有78.3%和87.7%的肽被裂解。sGnRH的最大降解发生在pH值为7至8的范围内。sGnRH和LHRH降解的主要初始产物是1-5、6-10和1-9片段。这表明涉及两种位点特异性肽酶，一种Tyr5-Gly6内肽酶和一种Pro9-Gly10NH2肽酶或脯氨酸后切割酶。虽然1-6和1-9片段经历快速的二次降解，但1-5相对稳定。竞争实验表明，在Tyr5-Gly6键处切割sGnRH的内肽酶对神经肽不具有特异性，可能是一种普通的蛋白水解酶。然而，在9-10键处的切割对sGnRH中发现的Pro9-Gly10NH2序列具有高度特异性。所提出的金头鲷的两种垂体肽酶具有一些与大鼠垂体和下丘脑GnRH切割酶相似的特征。在卵母细胞处于卵黄发生前期或卵黄发生后期的雌性之间，垂体GnRH降解活性没有差异。
