Electron Microscope Radioautography as a Quantitative Tool in Enzyme Cytochemistry

Abstract

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.

Normal distribution and denervation changes of neurotransmitter related enzymes in cholinergic neurones

“…A similar pre-and post-synaptic distribution is observed in the iris where practically all CAT (98 %) is in the ciliary nerve terminals TRANS-SYNAPTIC REGULATION OF CAT 243 and AChE is mostly concentrated in the muscle (80%). The 20% localization in the nerve terminal is similar to the AChE estimated for innervated vertebrate twitch muscle (Salpeter, 1967).…”

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

“…Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites//xm 2 of surface area of preand postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane (probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.KEY WORDS acetylcholinesterase extraocular muscle EM autoradiography track autoradiography neuromuscular junctions acetylcholine receptorIn several studies we have assessed the number of esterase sites both at the whole endplate (31, 32) and on the fine structural level (33,34,41). We found that in two different mouse muscles, the diaphragm and sternomastoid, there were about the same number of labeled sites per square micrometer of the folded postjunctional membrane (PJM).…”

“…In several studies we have assessed the number of esterase sites both at the whole endplate (31, 32) and on the fine structural level (33,34,41). We found that in two different mouse muscles, the diaphragm and sternomastoid, there were about the same number of labeled sites per square micrometer of the folded postjunctional membrane (PJM).…”

“…They were thoroughly washed in cold phosphate buffer with 6.8% sucrose, and then incubated either (a) in [~H]DFP, to label all DFP sites, or (b) with the sequence nonradioactive DFP ~ 2-PAM ~ [3H]DFP, to label only 2-PAM-reactivated sites. The incubation procedures were as previously described (33,34,41); DFP incubation was performed at pH 7.4 and 2-PAM incubation at pH 7.8, both in 0.06 M phosphate buffer. The tissues were then postfixed in 1% OsO4 in 0.06 M phosphate buffer, block stained in 2% aqueous uranyl nitrate or acetate, and embedded in Epon 812.…”

A quantitative study of cholinesterase in myoneural junctions from rat and guinea‐pig extraocular muscles

“…This is in good agreement with Barnard & Rogers (1967) who showed that in mouse extraocular muscles the number of cholinesterase molecules in twitch fibres was five times that in tonic fibres. This difference can be attributed to the less extensive synaptic folding in the myoneural junctions of the tonic fibres (Salpeter, 1967). It is unlikely that the different levels of cholinesterase activity are due to differing enzymes in fine and focal endings, since Silver (1963) and Dietert (1965) have shown that acetylthiocholine and butyrylthiocholine are hydrolysed similarly by the two types of ending.…”