Philpott C J, Rahman N A, Kenny N, Londo T R, Young R M, Barisas B G, Roess D A
Department of Physiology, Colorado State University, Fort Collins 80523, USA.
Biochim Biophys Acta. 1995 Apr 12;1235(1):62-8. doi: 10.1016/0005-2736(94)00277-v.
We have examined the molecular motions of luteinizing hormone (LH) receptor and the Major Histocompatibility Complex Class I antigen on murine Leydig cells. Using time-resolved phosphorescence anisotropy methods, erythrosin (ErITC)-derivatized ovine luteinizing hormone (oLH) bound to the LH receptor appears rotationally mobile with rotational correlation times of 19.6 +/- 1.3 microseconds, 13.3 +/- 2.4 microseconds, 9.5 +/- 0.7 microseconds and 4.7 +/- 0.5 microseconds at 4 degrees C, 15 degrees C, 25 degrees C and 37 degrees C, respectively. Rotational correlation times for human chorionic gonadotropin (hCG)-occupied LH receptors were similar to those of the ErITC-oLH occupied receptor at each temperature. In addition, both oLH- and hCG-occupied LH receptors were laterally mobile in fluorescence photobleaching recovery experiments with diffusion coefficients at 29 degrees C of (5.8 +/- 0.9) x 10(-10) cm2 s-1 and (2.9 +/- 0.4) x 10(-10) cm2 s-1, respectively. We also measured the rotational correlation time of Class I antigen on murine Leydig cells using ErITC-derivatized 34-12-2S, an anti-Class I monoclonal antibody. Because there was no decay of the anisotropy function at 4 degrees C, 15 degrees C, 25 degrees C or 37 degrees C in the absence of oLH or following preincubation of Leydig cells with 1 nM oLH, it appears that Class I is rotationally immobile on the 1 ms timescale of our experiments. This result is consistent with the presence of Class I antigen in large molecular weight structures and may be the result of Class I self-aggregation. Further, treatment of cells with anti-Class I antibody had no effect on either basal or oLH-stimulated testosterone secretion. Thus, it appears that this anti-Class I antibody is not LH-mimetic on murine Leydig cells.
我们研究了促黄体生成素（LH）受体和主要组织相容性复合体I类抗原在小鼠睾丸间质细胞上的分子运动。使用时间分辨磷光各向异性方法，与LH受体结合的赤藓红（ErITC）衍生化的绵羊促黄体生成素（oLH）在4℃、15℃、25℃和37℃时表现出旋转流动性，旋转相关时间分别为19.6±1.3微秒、13.3±2.4微秒、9.5±0.7微秒和4.7±0.5微秒。在每个温度下，人绒毛膜促性腺激素（hCG）占据的LH受体的旋转相关时间与ErITC - oLH占据的受体相似。此外，在荧光光漂白恢复实验中，oLH和hCG占据的LH受体都具有侧向流动性，在29℃时的扩散系数分别为(5.8±0.9)×10⁻¹⁰ cm² s⁻¹和(2.9±0.4)×10⁻¹⁰ cm² s⁻¹。我们还使用ErITC衍生化的抗I类单克隆抗体34 - 12 - 2S测量了小鼠睾丸间质细胞上I类抗原的旋转相关时间。因为在没有oLH的情况下，以及在睾丸间质细胞与1 nM oLH预孵育后，在4℃、15℃、25℃或37℃时各向异性函数没有衰减，所以在我们实验的1毫秒时间尺度上，I类似乎是旋转不动的。这一结果与I类抗原存在于大分子结构中一致，可能是I类自身聚集的结果。此外，用抗I类抗体处理细胞对基础或oLH刺激的睾酮分泌均无影响。因此，在小鼠睾丸间质细胞上，这种抗I类抗体似乎不具有LH模拟作用。
