Rubin B S, King J C, Millar R P, Seeburg P H, Arimura A
Endocrinology. 1987 Jul;121(1):305-9. doi: 10.1210/endo-121-1-305.
Results of previous immunocytochemical studies indicate that in the rat brain proteolytic cleaving of LHRH precursors to generate the physiologically active decapeptide takes place within neuronal fibers and terminals and not within perikarya. A 69-amino acid (aa) LHRH precursor comprised of the decapeptide, a 3-aa cleavage and amidation site, and a 56-aa C-terminal extension has recently been characterized. Two antisera generated to specific aa sequences of the C-terminal extension (RM 8/5, anti aa 14-26; PS 39A, anti aa 40-53) and two directed to specific regions of the LHRH decapeptide (RM 1076, anti aa 4-8; A 422 generated to the N-terminal pGlu and C-terminal amidated Gly) were used to further examine intraneuronal sites of precursor processing. Patterns of immunoreactivity revealed with antisera directed to non-LHRH sequences of LHRH precursor paralleled those observed with antisera to the decapeptide. Immunopositive perikarya, processes, and neurovascular terminals were observed with PS 39A. Antiserum PS 39A binds to an internal aa sequence of the C-terminal extension and would, therefore, be expected to detect intact precursor LHRH as well as products of proteolytic cleavage. In contrast, only immunopositive processes and neurovascular terminals were observed with RM 8/5, an antiserum directed to an initial aa sequence of the C-terminal extension. The pattern of immunoreactivity revealed with RM 8/5 resembled that observed with an antiserum that binds the fully processed decapeptide (A 422), indicating that proteolytic cleavage of the decapeptide from the C-terminal extension is required for binding by this antiserum. Furthermore, the restricted distribution of reaction product observed with RM 8/5 relative to A 422 suggests that additional processing of the C-terminal extension may be required for binding. Such additional processing appears to occur in neurovascular terminals of the median eminence.
以往免疫细胞化学研究结果表明，在大鼠脑中，促性腺激素释放激素（LHRH）前体经蛋白水解切割生成具有生理活性的十肽的过程发生在神经纤维和终末内，而非胞体内。最近已鉴定出一种由十肽、一个3氨基酸切割和酰胺化位点以及一个56氨基酸C末端延伸区组成的69个氨基酸（aa）的LHRH前体。针对C末端延伸区特定aa序列产生的两种抗血清（RM 8/5，抗aa 14 - 26；PS 39A，抗aa 40 - 53）以及针对LHRH十肽特定区域的两种抗血清（RM 1076，抗aa 4 - 8；针对N末端焦谷氨酸和C末端酰胺化甘氨酸产生的A 422）被用于进一步研究前体加工的神经元内位点。针对LHRH前体非LHRH序列的抗血清所揭示的免疫反应模式与针对十肽的抗血清所观察到的模式相似。用PS 39A观察到免疫阳性的胞体、突起和神经血管终末。抗血清PS 39A与C末端延伸区的一个内部aa序列结合，因此预期可检测完整的前体LHRH以及蛋白水解切割产物。相比之下，用RM 8/5（一种针对C末端延伸区起始aa序列的抗血清）仅观察到免疫阳性的突起和神经血管终末。RM 8/5所揭示的免疫反应模式类似于用结合完全加工的十肽的抗血清（A 422）所观察到的模式，表明该抗血清结合需要从C末端延伸区切割下十肽。此外，相对于A 422，RM 8/5观察到的反应产物分布受限表明结合可能需要C末端延伸区的额外加工。这种额外加工似乎发生在正中隆起的神经血管终末。
