利用钙激活发光蛋白奥贝林研究鸽红细胞“血影”对钙的通透性。

Permeability to calcium of pigeon erythrocyte 'ghosts' studied by using the calcium-activated luminescent protein, obelin.

作者信息

Campbell A K, Dormer R L

出版信息

Biochem J. 1975 Nov;152(2):255-65. doi: 10.1042/bj1520255.

DOI:10.1042/bj1520255

PMID:4062

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1172467/

Abstract

Obelin, the Ca(2+)-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ;ghosts' in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca(2+) within the ;ghosts' were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ;ghosts' during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ;ghosts'. Less than 10% of the inulin or pyruvate kinase sealed within the ;ghosts' was released under any of the experimental conditions. 3. Triton X-100 (0.1-10%, v/v) made the ;ghosts' highly permeable to Ca(2+). In the presence of 1mm-Ca(2+) and Triton, 95-100% of the obelin was utilized within 10-20s. 4. A time-course of resealing ;ghosts' at 37 degrees C showed that over a period of 90min, the ;ghosts' became gradually less permeable to Ca(2+). ;Ghosts' which remained at 0 degrees C retained only a small concentration of obelin and ATP, and were highly permeable to Ca(2+). 5. Erythrocyte ;ghosts' resealed for 30min at 20 degrees C rather than 37 degrees C were more permeable to Ca(2+), as shown by the fact that 92% of the obelin in the ;ghosts' was utilized during the first 60s after the addition of 1mm-Ca(2+), as opposed to 44% for ;ghosts' resealed at 37 degrees C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ;ghosts' which were highly permeable to Ca(2+) after resealing for 60min at 37 degrees C. Of the obelin in the ;ghosts', produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca(2+) compared with 23% for ;ghosts' produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ;ghosts' to Ca(2+). Maximum effects of the ionophore (16mug/ml) were obtained by preincubating the ;ghosts' with the ionophore A23187 (16mug/ml) in the presence of a low concentration of Mg(2+) and in the absence of Ca(2+).

摘要

将来自水螅水母的钙激活发光蛋白奥贝林密封在鸽红细胞“血影”内，以研究不同制备方法和二价阳离子载体A23187对其通透性的影响。2. 通过跟踪奥贝林的发光速率来研究“血影”内游离钙的变化。通过研究菊粉和丙酮酸激酶从“血影”中的释放情况，来探究实验过程中奥贝林可能从“血影”中释放出来的可能性。在任何实验条件下，密封在“血影”内的菊粉或丙酮酸激酶释放量均不到10%。3. 曲拉通X-100（0.1 - 10%，v/v）使“血影”对钙具有高度通透性。在1 mM钙和曲拉通存在的情况下，95 - 100%的奥贝林在10 - 20秒内被利用。4. 在37℃对“血影”进行重新封闭的时间进程表明，在90分钟的时间段内，“血影”对钙的通透性逐渐降低。保持在0℃的“血影”仅保留少量奥贝林和ATP，并且对钙具有高度通透性。5. 在20℃而非37℃重新封闭30分钟的红细胞“血影”对钙的通透性更高，这表现为加入1 mM钙后的最初60秒内，“血影”中92%的奥贝林被利用，而在37℃重新封闭的“血影”这一比例为44%。6. 在pH6.0而非pH7.0条件下溶血产生的“血影”，在37℃重新封闭60分钟后对钙具有高度通透性。pH6.0溶血产生的“血影”中的奥贝林，在加入1 mM钙后的最初60秒内90%被利用，而pH7.0产生的“血影”这一比例为23%。7. 二价阳离子载体A23187增加了“血影”对钙的通透性。在低浓度镁存在且无钙的情况下，用离子载体A23187（16 μg/ml）预孵育“血影”可获得该离子载体的最大效应（16 μg/ml）。