Free and bound acetylcholine in frog muscle.

1. Frog sartorius muscles were divided into end-plate containing (e.p.) and end-plate-free (non-e.p.) segments or homogenized in Ringer solution at 0 degrees C in the presence or absence of added acetylcholinesterase from electric eel. ACh was extracted from the tissue or from the homogenates and measured by mass fragmentography. 2. The concentration of ACh in non-e.p. segments was about six times lower than that in e.p. segments. 3. Homogenization of muscles in Ringer caused the hydrolysis of a small fraction ('free-1') of total ACh; addition of extra acetylcholinesterase caused hydrolysis of another, greater, fraction ('free-2' ACh). The esterase-resistant ('bound') ACh was stable at 0 degrees C up to 15 min of incubation. 4. Denervation for 15 days, which caused the disappearance of the nerve terminals, did not influence ACh in non-e.p. segments, but reduced total and bound ACh by about 75%, and free-2 ACh by 90%. 5. Treatment with La3+ ions, which caused the disappearance of synaptic vesicles, did not influence total ACh, but reduced bound ACh by 75%, whereas free-1 and free-2 ACh were increased. 6. Electrical stimulation of the nerve at 5 sec-1 or incubation with 50 mM-KCl did not affect ACh in the non-e.p. segments, but reduced by roughly 60% total, bound, and free ACh. 7. It is concluded that about 75% of bound ACh derives from synaptic vesicles, corresponding to 11,000 molecules per vesicle, and 25% from non-neural ACh; that free-1 and free-2 ACh derive mainly from the nerve terminal cytoplasm, although they may be contaminated by vesicular ACh.