An improved 45Ca protocol for investigating physiological mechanisms in coral calcification
An improved 45Ca protocol for investigating physiological mechanisms in coral calcification
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用于研究珊瑚钙化生理机制的改进 45Ca 协议
DOI：
10.1007/bf00350879
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发表时间：
1995
期刊：
影响因子：
2.4
通讯作者：
J. Jaubert
中科院分区：
文献类型：
--
作者：
É. Tambutté;É. Tambutté;D. Allemand;Isabelle Bourge;J. Gattuso;J. Jaubert
使用分枝石珊瑚 Stylophora pistillata 和 45Ca 的克隆珊瑚（以下简称“微菌落”）开发了一种敏感的实验方案，以便能够重复测量珊瑚钙化过程中涉及钙运输和分隔的生理和生化机制。克隆的 S. pistillata 小菌落是在实验室中利用 1990 年在约旦亚喀巴湾收集的亲本菌落的小碎片进行繁殖的。克隆微菌落具有几个有助于减少不必要的生物变异性的内在特性：（1）相同的基因型； (2) 尺寸、形状相似； (3) 不存在宏观钻探生物。通过产生没有骨骼表面暴露于放射性同位素标记的培养介质的微集落，进一步减少了与同位素交换相关的长期存在的问题相关的错误。该技术的价值主要在于其阐明运输途径和过程的卓越能力，而不在于其定量估计自然界珊瑚钙沉积的能力。我们在此描述了一种可快速交换的钙池，其中有高达 90% 的在孵化过程中吸收的放射性标记。该池（72.9±1.4 nmol Ca mg-1 蛋白）可能位于腔肠腔内，如下所示：（1）它具有 4 分钟半时饱和动力学； (2)钙的积累与海水中的钙浓度呈线性相关； (3)它对代谢和离子转运抑制剂不敏感表明膜不能隔离该隔室。与文献中描述的先前 45Ca2+ 方法相比，变异系数减少了 10% 至 40%，证明了这一大型细胞外池的冲洗大大改善了钙沉积的估计。使用碱度异常技术和此处描述的 45Ca 协议同时进行的钙化测量的比较表明，两种技术的相关系数接近 1。与以前的报告不同，我们的 45Ca2+ 衍生测量值略低于通过碱度消耗技术计算的测量值。
A sensitive experimental protocol using cloned corals (hereafter “microcolonies”) of the branching scleractinian coral Stylophora pistillata and 45Ca has been developed to enable reproducible measurements of physiological and biochemical mechanisms involved in calcium transport and compartmentalization during coral calcification. Cloned S. pistillata microcolonies were propagated in the laboratory from small fragments of parent colonies collected in 1990 in the Gulf of Aqaba, Jordan. Cloned microcolonies have several intrinsic properties that help to reduce unwanted biological variability: (1) same genotype; (2) similar sizes and shapes; and (3) absence of macroscopic boring organisms. Errors specifically associated with long-standing problems to do with isotopic exchange were further reduced by producing microcolonies with no skeletal surfaces exposed to the radioisotope-labelled incubation medium. The value of the technique resides principally in its superior ability to elucidate transportation pathways and processes and not in its ability to quantitatively estimate calcium deposition by corals in nature. We describe here a rapidly exchangeable calcium pool in which up to 90% of the radioactive label taken up during incubations is located. This pool (72.9±1.4 nmol Ca mg-1 protein) is presumably located within the coelenteric cavity as suggested by the following: (1) it has 4-min half-time saturation kinetics; (2) the accumulation of calcium is linearly correlated with the calcium concentration of sea-water; and (3) its insensitivity to metabolic and ion transport inhibitors indicate that membranes do not isolate this compartment. Washout of this large extracellular pool greatly improved estimates of calcium deposition as evidenced by 10 to 40% reduction in coefficients of variation when compared with previous 45Ca2+ methods described in the literature. Comparisons of calcification measurements simultaneously carried out using the alkalinity anomaly technique and the 45Ca protocol described here show that the correlation coefficient of both techniques is close to 1. Unlike previous reports, our 45Ca2+-derived measurements are slightly lower than those computed from the alkalinity depletion technique.