Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore.
Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore.
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DOI：
10.1016/s0021-9258(18)32420-7
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发表时间：
1983-05
期刊：
影响因子：
--
通讯作者：
D. Babcock
中科院分区：
文献类型：
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作者：
D. Babcock
荧光素和carboxyfluorescein发现最近的应用程序作为探针的细胞内pH值。本研究探讨了几个参数所需的光谱信息的解释来自荧光素和carboxyfluorescein产生的细胞内从他们的渗透二乙酸衍生物。系数测定的pH值依赖性的差异吸光度，吸光度比，和荧光发射强度比在选定的波长对羧基荧光素在水性缓冲液。评估了光散射对稀细胞悬浮液中羧基荧光素报告的表观pH的影响。一个明显的细胞内酸化与增加内部染料浓度被发现可能是由于细胞内探针与自身的相互作用。在这些考虑因素所施加的实验限制内工作，利用吸光度或荧光的直接测量或在释放内部羧基荧光素时观察到的零光谱响应的测定的方案都表明牛附睾精子的胞质空间维持在pH 6.5 - 6.6。单价阳离子特异性的羧酸离子载体，尼日利亚菌素和莫能菌素，被用来在细胞中产生跨膜质子梯度，使其在初步孵育中产生细胞内羧基荧光素，然后重新悬浮在介质中缓冲在相同的pH值作为精子胞质溶胶，但不同的阳离子组合物。通过插值至零响应，牛精子中的初始内部Na+和K+浓度分别估计为14 +/-2和120 +/-5 mM。质子或一价阳离子的初始跨膜梯度的能力，以促进等效的内部pH值的变化后，离子载体除了精子悬浮液支持一个简单的模型，预测稳态阳离子分布的应用。假设细胞溶质质子缓冲液的pKa接近测定的内部pH，这些实验允许额外计算该缓冲液在牛精子中的浓度为190 +/-20 meq/L。
Fluorescein and carboxyfluorescein have found recent application as probes of intracellular pH. The present study examines several parameters required for interpretation of the spectral information derived from fluorescein and carboxyfluorescein generated intracellularly from their permeant diacetate derivatives. Coefficients were determined for the pH dependence of the difference absorbance, of the absorbance ratios, and of the fluorescence emission intensity ratios at selected wavelength pairs for carboxyfluorescein in aqueous buffers. The effect of light scattering on the apparent pH reported by carboxyfluorescein in dilute cell suspensions was assessed. An apparent intracellular acidification associated with increasing internal dye concentration was found to result probably from interactions of the intracellular probe with itself. Working within the experimental limitations imposed by these considerations, protocols utilizing either direct measurement of absorbance or fluorescence or determination of the null spectral response observed upon release of internal carboxyfluorescein all indicate that the cytosolic space of bovine epididymal sperm is maintained at pH 6.5-6.6. The monovalent-cation-specific, carboxylic acid ionophores, nigericin and monensin, were utilized to produce transmembrane proton gradients in cells that were allowed to generate intracellular carboxyfluorescein in a preliminary incubation, then resuspended in media buffered at the same pH as the sperm cytosol but of varying cation composition. By interpolation to the null response, the initial internal Na+ and K+ concentrations in bovine sperm were estimated as 14 +/- 2 and 120 +/- 5 mM, respectively. The ability of initial transmembrane gradients of either protons or of monovalent cations to promote equivalent changes in internal pH following ionophore addition to sperm suspensions supports application of a simple model that predicts steady state cation distributions. With the assumption that the cytosolic proton buffer has a pKa near the determined internal pH, these experiments allow the additional calculation that this buffer is present at a concentration of 190 +/- 20 meq/liter in the bovine sperm.