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本工作对大鼠肾及小鼠十二指肠等进行了光学显微镜下腺苷环化酶的细胞化学研究;并在大鼠肾、颌下腺及肝等组织进行了电镜细胞化学定位的观察。采用 ATP 和特异性底物AMP-PNP 显示腺苷环化酶的活性。实验结果表明:(1)温育在含有和不含有底物的孵育液中,电镜下均观察到在组织的相同部位呈现出黑色电子密度沉淀,这种沉淀物的出现对戊二醛固定较为敏感。光学显微镜下,经不加底物的孵育液温育的切片均为阴性反应。(2)孵育液内加入腺苷环化酶的激活剂(氟化钠)或抑制剂(四氧嘧啶),其反应沉淀物的出现均无明显变化。(3)在碱性磷酸酶含量较高的组织(肾、十二指肠)中,以 AMP-PNP 为底物,在光学显微镜下观察到有碱性磷酸酶反应的干扰。(4)组织小块或冰冻切片经煮沸加热处理后,在光学显微镜和电镜下均不出现沉淀物。我们的实验结果进一步对 R-H 氏显示腺苷环化酶的细胞化学方法的有效估价表示怀疑。
In this work, rat kidney and mouse duodenum and other light microscopy of adenylyl cyclase cytochemistry; and in rat kidney, submandibular gland and liver tissue electron microscopy cytochemical localization was observed. ATP and specific substrate AMP-PNP showed adenosine cyclase activity. The experimental results showed that: (1) Incubation with and without substrate incubated in electron microscope showed the presence of black electron density precipitation in the same part of the tissue, the appearance of this precipitate on glutaraldehyde is relatively fixed sensitive. Under the light microscope, the sections incubated with the substrate-free incubation solution were negative. (2) The addition of adenosine cyclase activator (sodium fluoride) or inhibitor (alloxan) to the incubation solution showed no significant change in the reaction precipitate. (3) In alkaline phosphatase (kidney, duodenum), AMP-PNP was used as substrate to observe the interference of alkaline phosphatase under light microscope. (4) Tissue pieces or frozen sections were boiled and heat-treated, and no sediment appeared under light microscope or electron microscope. Our experimental results further cast doubt on the effective valuation of R-H’s cytochemical methods of displaying adenylyl cyclase.