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用标记抑制剂法对横纹肌结构中的酶进行定量研究。II. 通过液体闪烁计数法对放射自显影测量的确认。

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting.

作者信息

Rogers A W, Barnard E A

出版信息

J Cell Biol. 1969 Jun;41(3):686-95. doi: 10.1083/jcb.41.3.686.

DOI:10.1083/jcb.41.3.686
PMID:5768869
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2107826/
Abstract

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-(3)H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10(7)molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.

摘要

将小鼠膈肌和胸锁乳突肌的碎片在已知能使所有可用的二异丙基氟磷酸酯（DFP）-（3）H敏感反应位点饱和的条件下进行孵育。经过充分洗涤后，用吡啶-2-醛肟甲碘化物（2-PAM）处理使乙酰胆碱酯酶（AChase）特异性重新激活。通过液体闪烁计数测量2-PAM释放到溶液中的放射性DP基团，并将其与存在的运动终板的已知数量相关联。在将过量的吸附放射性降低到可接受水平时遇到了相当大的困难：需要长时间的洗涤程序、用有机溶剂萃取以及通过显微解剖去除多余的肌纤维。六个实验得出每个胸锁乳突肌终板的AChase分子平均值为2.4×10（7），这与先前通过放射自显影报道的测量结果相当一致。

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Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting.用标记抑制剂法对横纹肌结构中的酶进行定量研究。II. 通过液体闪烁计数法对放射自显影测量的确认。
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引用本文的文献

1
The labelled inhibitor method in cytoenzymology.细胞酶学中的标记抑制剂法。
Histochem J. 1972 Sep;4(5):467-76. doi: 10.1007/BF01012534.
2
Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.用标记抑制剂法对横纹肌结构中的酶进行定量研究。I. 通过放射自显影法测定运动终板处乙酰胆碱酯酶分子及其他与二异丙基氟磷酸（DFP）反应位点的数量。
J Cell Biol. 1969 Jun;41(3):665-85. doi: 10.1083/jcb.41.3.665.

本文引用的文献

1
A histochemical method for localizing cholinesterase activity.一种用于定位胆碱酯酶活性的组织化学方法。
Proc Soc Exp Biol Med. 1949 Apr;70(4):617-22. doi: 10.3181/00379727-70-17013.
2
[Inhibition and reactivation of cholinesterases after poisoning with paraoxon and DFP in vitro].[对氧磷和二异丙基氟磷酸酯体外中毒后胆碱酯酶的抑制与复活]
Naunyn Schmiedebergs Arch Exp Pathol Pharmakol. 1961;240:514-22.
3
Molecular complementariness as basis for reactivation of alkyl phosphate-inhibited enzyme.以分子互补性作为磷酸烷基酯抑制酶再激活的基础。
Arch Biochem Biophys. 1958 Oct;77(2):286-96. doi: 10.1016/0003-9861(58)90077-8.
4
Chemical reactivation of phosphorylated human and bovine true cholinesterases.磷酸化人及牛真性胆碱酯酶的化学再活化
Br J Pharmacol Chemother. 1956 Sep;11(3):295-303. doi: 10.1111/j.1476-5381.1956.tb01069.x.
5
Evaluation of quench correction in liquid scintillation counting by internal, automatic external, and channels' ratio standardization methods.
Anal Biochem. 1966 Aug;16(2):206-19. doi: 10.1016/0003-2697(66)90148-5.
6
Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.用标记抑制剂法对横纹肌结构中的酶进行定量研究。I. 通过放射自显影法测定运动终板处乙酰胆碱酯酶分子及其他与二异丙基氟磷酸（DFP）反应位点的数量。
J Cell Biol. 1969 Jun;41(3):665-85. doi: 10.1083/jcb.41.3.665.

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