A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY I. The Distribution of Acetylcholinesterase at Motor End Plates of a Vertebrate Twitch Muscle

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85 %) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region. The problem of the localization and quantitation of acetylcholinesterase at neuromuscular junctions is an old one and has been studied in a variety of ways both biochemically and histochemically. (Review of the early literature is given in Couteaux 1955; Koelle, 1963.) Most studies favored the view that the enzyme is located primarily in the subneural apparatus. A notable exception was expressed by Nachmansohn (1963). Recently, the problem of localization has been reopened with the use of electron microscope histochemical techniques (Lehrer and Ornstein, 1959; Barrnett, 1961; Zacks and Blumberg, 1961; Miledi, 1964) and the problem of quantitation has been attacked with the use of irreversible enzyme inhibitors (Ostrowski and Barnard, 1961; Barnard and Ostrowski, 1964; Waser and Relier, 1965). The use of radioautography has been suggested by Ostrowski and Barnard (1961) for the localization of enzyme sites that had been labeled with radioactive inhibitors. These investigators point out that radioautography could be used for the determination of sites of some enzymes for which color reactions are notavailable; that radioautography provides a valuable alternative to, and check on, enzyme localization by color reactions, and that radioautography with enzyme inhibitors lends itself to quantitative evaluation. These authors applied light microscope radioautography to the quantitative evaluation of acetylcholinesterase (AChase) at motor end plates using the irreversible enzyme inhibitor diisopropylfluorophosphate-~H (DFP-3H) (Ostrowski et al., 1963; Barnard and Ostrowski, 1964). It has subsequently been shown (Rogers et al., 1966) that some quantitative results reported in these inves379 on M ay 0, 2017 D ow nladed fom Published February 1, 1967