QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS II. Confirmation of Radioautographic Measurement by Liquid-Scintillation Counting

Rogers , A. W.; Barnard , E. A.

doi:10.1083/jcb.41.3.686

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Article| June 01 1969

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : II. Confirmation of Radioautographic Measurement by Liquid-Scintillation Counting

A. W. Rogers,

A. W. Rogers

From the Molecular Enzymology Unit, Departments of Biochemistry and Biochemical Pharmacology, State University of New York, Buffalo, New York 14214.

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E. A. Barnard

From the Molecular Enzymology Unit, Departments of Biochemistry and Biochemical Pharmacology, State University of New York, Buffalo, New York 14214.

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Author and Article Information

A. W. Rogers

From the Molecular Enzymology Unit, Departments of Biochemistry and Biochemical Pharmacology, State University of New York, Buffalo, New York 14214.

E. A. Barnard

From the Molecular Enzymology Unit, Departments of Biochemistry and Biochemical Pharmacology, State University of New York, Buffalo, New York 14214.

Dr. Rogers' present address is M.R.C. Neuroendocrinology Research Unit, Department of Human Anatomy, Oxford, England. Reprint requests and inquiries should be addressed to Dr. E. A. Barnard, Molecular Enzymology Unit, State University of New York, Buffalo, N. Y. 14214

Received: November 28 1967

Revision Received: December 09 1968

Online ISSN: 1540-8140

Print ISSN: 0021-9525

1969

J Cell Biol (1969) 41 (3): 686–695.

https://doi.org/10.1083/jcb.41.3.686

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-³H 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 10⁷molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.

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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

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