Nokubo M, Nagy I, Kitani K, Ohta M
First Laboratory of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Japan.
Biochim Biophys Acta. 1988 Apr 22;939(3):441-8. doi: 10.1016/0005-2736(88)90090-9.
The autofluorescence of isolated rat liver cell plasma membranes was characterized in vitro in relation to the autofluorescence used previously for fluorescence recovery after photobleaching (FRAP) studies. The fluorescence of membrane preparations displayed an emission pattern with a maximum at around 525 nm when excited with a 468 nm blue light. The excitation spectrum monitored at 525 nm closely resembled that of flavin compounds (riboflavin, FAD, FMN). The chloroform extract of the membrane fraction showed practically no fluorescence, whereas, both the water-soluble and water-insoluble protein fractions remaining after chloroform extraction were strongly fluorescent. The fluorescence disappeared almost completely under the effect of sodium hydrosulfite, and recovered after oxidation either by shaking in air or by adding buffered hydrogen peroxide solution. The fluorescence of the acid extract of the plasma membranes photolyzed in an alkaline medium was quite similar to that of lumiflavin obtained from the photolysis of riboflavin in an alkaline medium. The plasma membranes prepared from isolated hepatocytes (which were completely devoid of endothelial cell contamination) exhibited the same autofluorescence in the liver cell plasma membranes. The results suggest that the autofluorescence of the liver cell plasma membranes is most likely of a character similar to that of flavin, bound to hepatocyte plasma membrane proteins. This fluorescence is suitable for measuring the average lateral diffusion constant of proteins by means of FRAP methods.
在体外对分离的大鼠肝细胞质膜的自发荧光进行了表征，并将其与先前用于光漂白后荧光恢复（FRAP）研究的自发荧光相关联。当用468nm蓝光激发时，膜制剂的荧光显示出发射模式，其最大值在525nm左右。在525nm处监测的激发光谱与黄素化合物（核黄素、FAD、FMN）的激发光谱非常相似。膜部分的氯仿提取物几乎没有荧光，而氯仿提取后剩余的水溶性和水不溶性蛋白质部分都有强烈的荧光。在亚硫酸氢钠的作用下，荧光几乎完全消失，在空气中振荡或加入缓冲过氧化氢溶液氧化后恢复。在碱性介质中光解的质膜酸提取物的荧光与在碱性介质中核黄素光解得到的黄素荧光非常相似。从分离的肝细胞（完全没有内皮细胞污染）制备的质膜在肝细胞质膜中表现出相同的自发荧光。结果表明，肝细胞质膜的自发荧光很可能具有与结合在肝细胞质膜蛋白上的黄素相似的特征。这种荧光适用于通过FRAP方法测量蛋白质的平均横向扩散常数。
