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Enzyme histochemistry of the retina at light activation.

光激活时视网膜的酶组织化学。

基本信息

批准号：
60480101
负责人：
SATITO T.
金额：
$ 4.29万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

The development of the rapid freeze substitution method enables us to see excellent morphology in electron microscopy. Therefore, the possibility of enzyme histochemistry of cyclic nucleotide metabolizing enzymes on the quick feeze substituted rat retina was studied.The ultrastructure of the freeze substituted samples was excellent in showing the fine parallel arrangement of the disk membranes in the rod outer segnt.In spite of a long substitution fixation of 3 days, using 0.5% glutraldehyde and 6% tannic acid in acetone, guanylate cyclase, phosphodiesterase, CTPst, and 5'-nucleotidase were demonstrably retained. With the contrast enhancemen on biogical membranes by the 0.5-4% glutaraldehyde and 6% tannic acid in aceton, the site of phosphodiesterase activity was clearly visualized on the disk membranes of rod outer segment sepecially at cytoplasmic side. The low phosphodiesterase activity was observed in the rod outer segment from the dark adapted retina showing as some disk membranes were positive but the others were not. All disk membranes were appleared positive and enhanced at light conditions.The present investigation reveals that the freeze substitution technique can preserve not only fine morphology but also enzyme activity in cells and tissues at cerain physiological conditions.

快速冷冻置换法的发展使我们能够在电子显微镜中看到优异的形态。本文研究了快速冷冻替代大鼠视网膜环核苷酸代谢酶的酶组织化学的可能性，冷冻替代样品的超微结构良好，显示了棒外段盘膜的精细平行排列，尽管用0.5%戊二醛和6%鞣酸的丙酮溶液进行长时间的替代固定，鸟苷酸环化酶，磷酸二酯酶，CTPst和5 '-核苷酸酶明显保留。用0.5-4%戊二醛和6%单宁酸的丙酮溶液对细胞膜进行对比增强，可清楚地观察到磷酸二酯酶活性部位在杆状细胞外节的盘膜上，尤其是胞质侧。暗适应视网膜视杆外段磷酸二酯酶活性较低，表现为部分盘膜呈阳性，而另一些则不呈阳性。所有盘膜均呈苹果样阳性，并在光照条件下增强.本研究表明，冷冻置换技术在一定的生理条件下不仅能保持细胞和组织的良好形态，而且能保持酶的活性.