Electron Microscope Radioautography as a Quantitative Tool in Enzyme Cytochemistry

Abstract

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP'H . Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per a' of cleft tissue or 12,000 sites per µ2 of postjunctional membrane surface area . A considerable concentration is also present in the teloglial cap . It has previously been demonstrated ) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)-a compound which selectively reactivates phosphorylated acetylcholinesterase . In the present study, it was found that this ratio of 1 :2 holds also on a fine-structural level . Muscle mast cells were found to have a heavy concentration of bound DFP.

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.…”

“…The muscles were either fixed in vitro by immersion in 1.5 % glutaraldehyde in 0.06 M phosphate buffer, pH 7.4 (2 h on ice as previously done [34,41]), or by perfusion of the whole animal in the periodate lysineparaformaldehyde fixative (2% in 0.06 M phosphate with 6.8% sucrose) (27), followed by 2-h immersion fixation in the same fixatives. In a recent study,' it was found that the DFP-binding-site density is not significantly different with these two fixation procedures.…”

“…Once this was done, all animals were analyzed for absolute quantitation. Grain density (grains per unit area or volume of each potential source) was .tabulated, and the absolute site density calculated as previously described 2 (34,37,41).…”