Radiochemical assay for ACh: modifications for sub‐picomole measurements

This procedure involves the conversion of ACh to choline-32P04 in the presence of AT32P and the enzymes, acetylcholinesterase and choline kinase (ATP: choline phosphotransferase, EC 2.7.1.32). With this procedure levels of ACh in mammalian brain have been determined in as little as 200pg of wet tissue. In this laboratory, the assay has been used primarily for the determination of endogenous levels of ACh in individual soma isolated from the CNS of Aplysia californica (MCCAMAN et al., 1973). ACh was measurable in only a few identified cells within certain ganglia and among these particular cells, the ACh content was proportional to the cell volume (the average concentration was calculated to be about 0 .5m~) . Since the sensitivity of the procedure was approx 2.5 pmol, this limited the study of individual soma to those that were 200pm or larger in diameter; such soma comprise less than 0.1% of the total number per ganglion. The present report describes recent modifications of the assay which have resulted in a substantial increase in sensitivity (to 0.04 pmol), primarily by increasing the specific activity of the [y-”P]ATP and decreasing the reagent blank. Values for ACh are proportional to tissue over a 500-fold range, from approx 0.1-50 pg protein. Comparison of ACh content of individual soma with both procedures is also given. BERG & MCCAMAN, 1973; GOLDBERC & MCCAMAN, 1974).