Lew R A, Tetaz T J, Glucksman M J, Roberts J L, Smith A I
Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
J Biol Chem. 1994 Apr 29;269(17):12626-32.
The metalloendopeptidase EC 3.4.24.15 is believed to degrade gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) by cleavage at the Tyr5-Gly6 bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine hypothalamus with recombinant rat testicular endopeptidase 24.15 to degrade synthetic GnRH. Both soluble and membrane hypothalamic fractions degraded GnRH to GnRH1-5, with some production of GnRH1-9 and GnRH1-3. Generation of the smaller fragments was blocked by a specific endopeptidase 24.15 inhibitor (CFP-AAY-pAB), but production of GnRH1-9 was reciprocally enhanced, suggesting this peptide may be an intermediate generated by prolyl endopeptidase. Indeed, both bacitracin and Z-Pro-prolinal, inhibitors of this enzyme, markedly reduced GnRH degradation to any product. Degradation of synthetic GnRH1-9 was more rapid than that of GnRH and was inhibited by CFP-AAY-pAB but not bacitracin. Activity against either substrate was greater in the soluble fraction. Repeated washing of the membrane fraction followed by extraction with Triton X-114 suggested that both endopeptidase 24.15 and prolyl endopeptidase, although predominantly soluble, can be peripherally associated with membranes. When fractionated by hydrophobic interaction chromatography, soluble endopeptidase 24.15 degraded GnRH only in fractions that also exhibited prolyl endopeptidase activity. In contrast, maximal degradation of GnRH1-9 was observed in adjacent fractions, which also contained the highest levels of immunoreactive endopeptidase 24.15. The affinity of recombinant endopeptidase 24.15 for GnRH was low (Km = 1.35 mM), was improved 10-15-fold by removal of the COOH-terminal amide or glycinamide (Km = 90 and 119 microM, respectively), and could be inhibited by CFP-AAY-pAB but not bacitracin. Taken together, these results suggest that GnRH metabolism in the hypothalamus may occur via a two-step process involving first removal of Gly10-NH2 by prolyl endopeptidase, followed by cleavage by endopeptidase 24.15 at the Tyr5-Gly6 bond.
金属内肽酶EC 3.4.24.15被认为通过在Tyr5-Gly6键处切割来降解促性腺激素释放激素（GnRH）（焦谷氨酸-组氨酸-色氨酸-丝氨酸-酪氨酸-甘氨酸-亮氨酸-精氨酸-脯氨酸-甘氨酸-酰胺）。我们比较了来自绵羊下丘脑的粗制和部分纯化的内肽酶24.15与重组大鼠睾丸内肽酶24.15降解合成GnRH的能力。下丘脑的可溶性和膜性部分都将GnRH降解为GnRH1-5，同时产生了一些GnRH1-9和GnRH1-3。较小片段的产生被特异性内肽酶24.15抑制剂（CFP-AAY-pAB）阻断，但GnRH1-9的产生反而增加，这表明该肽可能是脯氨酰内肽酶产生的中间体。事实上，杆菌肽和Z-脯氨酰醛，这种酶的抑制剂，都显著减少了GnRH降解为任何产物。合成GnRH1-9的降解比GnRH更快，并且被CFP-AAY-pAB抑制，但不被杆菌肽抑制。可溶性部分对任何一种底物的活性都更高。用Triton X-114对膜性部分进行反复洗涤然后提取表明，内肽酶24.15和脯氨酰内肽酶虽然主要是可溶性的，但也可以与膜外周结合。当通过疏水相互作用色谱法分级分离时，可溶性内肽酶24.15仅在也表现出脯氨酰内肽酶活性的级分中降解GnRH。相反，在相邻级分中观察到GnRH1-9的最大降解，这些级分也含有最高水平的免疫反应性内肽酶24.15。重组内肽酶24.15对GnRH的亲和力较低（Km = 1.35 mM），通过去除COOH末端酰胺或甘氨酰胺（分别为Km = 90和119 microM）可提高10-15倍，并且可被CFP-AAY-pAB抑制，但不被杆菌肽抑制。综上所述这些结果表明，下丘脑GnRH代谢可能通过两步过程发生，首先由脯氨酰内肽酶去除Gly10-NH2，然后由内肽酶24.15在Tyr5-Gly6键处切割。
