Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1969 Jun 1;41(3):665–685. doi: 10.1083/jcb.41.3.665

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

A W Rogers 1, Z Darzynkiewicz 1, M M Salpeter 1, K Ostrowski 1, E A Barnard 1
PMCID: PMC2107829  PMID: 4181315

Abstract

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-32P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 107 sites per endplate, in the diaphragm, about 3 x 107. Reaction with DFP-32P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 107 and 1.1 x 107per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AUSTIN L., BERRY W. K. Two selective inhibitors of cholinesterase. Biochem J. 1953 Jul;54(4):695–700. [PMC free article] [PubMed] [Google Scholar]
  2. BARNARD E. A., MARBROOK J. Quantitative cytochemistry using directly applied radioactive reagents. Nature. 1961 Feb 4;189:412–413. doi: 10.1038/189412a0. [DOI] [PubMed] [Google Scholar]
  3. BAYLISS B. J., TODRICK A. The use of a selective acetylcholinesterase inhibitor in the estimation of pseudocholinesterase activity in rat brain. Biochem J. 1956 Jan;62(1):62–67. doi: 10.1042/bj0620062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Changeux J. P. Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol Pharmacol. 1966 Sep;2(5):369–392. [PubMed] [Google Scholar]
  5. DAVIES D. R., GREEN A. L. The kinetics of reactivation, by oximes, of cholinesterase inhibited by organophosphorus compounds. Biochem J. 1956 Aug;63(4):529–535. doi: 10.1042/bj0630529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DENZ F. A. On the histochemistry of the myoneural junction. Br J Exp Pathol. 1953 Jun;34(3):329–339. [PMC free article] [PubMed] [Google Scholar]
  7. Darzynkiewicz Z., Rogers A. W., Barnard E. A. Quantitative analysis of the esterases in rat megakaryocytes by a radio-isotope cytochemical technique. J Histochem Cytochem. 1966 May;14(5):379–384. doi: 10.1177/14.5.379. [DOI] [PubMed] [Google Scholar]
  8. Darzynkiewicz Z., Rogers A. W., Barnard E. A., Wang D. H., Werkheiser W. C. Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase. Science. 1966 Mar 25;151(3717):1528–1530. doi: 10.1126/science.151.3717.1528. [DOI] [PubMed] [Google Scholar]
  9. Darzynkiewicz Z, Rogers A W, Barnard E A. The number of acetylcholinesterase molecules in the rat megakaryocyte. J Histochem Cytochem. 1966 Dec;14(12):915–922. doi: 10.1177/14.12.915. [DOI] [PubMed] [Google Scholar]
  10. FALK G. J., KING R. C. RADIOAUTOGRAPHIC EFFICIENCY FOR TRITIUM AS A FUNCTION OF SECTION THICKNESS. Radiat Res. 1963 Nov;20:466–470. [PubMed] [Google Scholar]
  11. FULTON M. P., MOGEY G. A. Some selective inhibitors of true cholinesterase. Br J Pharmacol Chemother. 1954 Jun;9(2):138–144. doi: 10.1111/j.1476-5381.1954.tb00832.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GIACOBINI E., HOLMSTEDT B. Cholinesterase in muscles: a histochemical and microgasometric study. Acta Pharmacol Toxicol (Copenh) 1960;17:94–105. doi: 10.1111/j.1600-0773.1960.tb01233.x. [DOI] [PubMed] [Google Scholar]
  13. GUIDOTTI G. G., SPINELLI-RESSI F. INCORPORATION OF SULPHATE IN PERITONEAL MAST CELLS ISOLATED FROM YOUNG AND ADULT RATS. Exp Cell Res. 1964 Jan;33:254–263. doi: 10.1016/s0014-4827(64)81031-4. [DOI] [PubMed] [Google Scholar]
  14. HOBBIGER F. Chemical reactivation of phosphorylated human and bovine true cholinesterases. Br J Pharmacol Chemother. 1956 Sep;11(3):295–303. doi: 10.1111/j.1476-5381.1956.tb01069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HOLMSTEDT B. A modification of the thiocholine method for the determination of cholinesterase. I. Biochemical evaluation of selective inhibitors. Acta Physiol Scand. 1957 Oct 22;40(4):322–330. doi: 10.1111/j.1748-1716.1957.tb01500.x. [DOI] [PubMed] [Google Scholar]
  16. HOPSU V. K., PONTINEN J. THE MOTOR END-PLATE REACTION WITH NAPHTHOL-AS EPSILON-AMINOCAPROATE. J Histochem Cytochem. 1964 Nov;12:853–854. doi: 10.1177/12.11.853. [DOI] [PubMed] [Google Scholar]
  17. KARNOVSKY M. J., ROOTS L. A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. J Histochem Cytochem. 1964 Mar;12:219–221. doi: 10.1177/12.3.219. [DOI] [PubMed] [Google Scholar]
  18. KISIELESKI W. E., BASERGA R., VAUPOTIC J. The correlation of autoradiographic grain counts and tritium concentration in tissue sections containing tritiated thymidine. Radiat Res. 1961 Sep;15:341–348. [PubMed] [Google Scholar]
  19. KOELLE G. B., FRIEDENWALD J. A. A histochemical method for localizing cholinesterase activity. Proc Soc Exp Biol Med. 1949 Apr;70(4):617–622. doi: 10.3181/00379727-70-17013. [DOI] [PubMed] [Google Scholar]
  20. KREMZNER L. T., WILSON I. B. A PARTIAL CHARACTERIZATION OF ACETYLCHOLINESTERASE. Biochemistry. 1964 Dec;3:1902–1905. doi: 10.1021/bi00900a020. [DOI] [PubMed] [Google Scholar]
  21. LATKI O., ERDMANN W. D. [Inhibition and reactivation of cholinesterases after poisoning with paraoxon and DFP in vitro]. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol. 1961;240:514–522. [PubMed] [Google Scholar]
  22. LEVINTHAL C., THOMAS C. A., Jr Molecular autoradiography: the beta-ray counting from single virus particles and DNA molecules in nuclear emulsions. Biochim Biophys Acta. 1957 Mar;23(3):453–465. doi: 10.1016/0006-3002(57)90363-3. [DOI] [PubMed] [Google Scholar]
  23. OSTROWSKI K., BARNARD E. A. Application of isotopically labelled specific inhibitors as a method in enzyme cytochemistry. Exp Cell Res. 1961 Nov;25:465–468. doi: 10.1016/0014-4827(61)90298-1. [DOI] [PubMed] [Google Scholar]
  24. OSTROWSKI K., BARNARD E. A., STOCKA Z., DARZYNKIEWICZ Z. AUTORADIOGRAPHIC METHODS IN ENZYME CYTOCHEMISTRY. I. LOCALISATION OF ACETYLCHOLINESTERASE ACTIVITY USING A 3-H LABELED IRREVERSIBLE INHIBITOR. Exp Cell Res. 1963 Jun;31:89–99. doi: 10.1016/0014-4827(63)90158-7. [DOI] [PubMed] [Google Scholar]
  25. Rogers A. W., Barnard E. A. Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting. J Cell Biol. 1969 Jun;41(3):686–695. doi: 10.1083/jcb.41.3.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rogers A. W., Darzynkiewicz Z., Barnard E. A., Salpeter M. M. Number and location of acetylcholinesterase molecules at motor endplates of the mouse. Nature. 1966 Jun 4;210(5040):1003–1006. doi: 10.1038/2101003a0. [DOI] [PubMed] [Google Scholar]
  27. Salpeter M. M. Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle. J Cell Biol. 1967 Feb;32(2):379–389. doi: 10.1083/jcb.32.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. TODRICK A. The inhibition of cholinesterases by antagonists of acetylcholine and histamine. Br J Pharmacol Chemother. 1954 Mar;9(1):76–83. doi: 10.1111/j.1476-5381.1954.tb00820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. WILSON I. B., GINSBURG B. A powerful reactivator of alkylphosphate-inhibited acetylcholinesterase. Biochim Biophys Acta. 1955 Sep;18(1):168–170. doi: 10.1016/0006-3002(55)90040-8. [DOI] [PubMed] [Google Scholar]
  30. WILSON I. B., GINSBURG S., QUAN C. Molecular complementariness as basis for reactivation of alkyl phosphate-inhibited enzyme. Arch Biochem Biophys. 1958 Oct;77(2):286–296. doi: 10.1016/0003-9861(58)90077-8. [DOI] [PubMed] [Google Scholar]
  31. WILSON I. B., HARRISON M. A., GINSBURG S. Carbamyl derivatives of acetylcholinesterase. J Biol Chem. 1961 May;236:1498–1500. [PubMed] [Google Scholar]
  32. Waser P. G., Reller J. Bestimmung der Zahl aktiver Zentren der Acetylcholinesterase in motorischen Endplatten. Experientia. 1965 Jul 15;21(7):402–403. doi: 10.1007/BF02139769. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES