Choline Acetyltransferase from Rat Brain

Abstract

Abstract The localization, purification, and some enzymatic properties of choline acetyltransferase from rat brain were studied. Most assays were performed with a specific radiometric micromethod. Solubilization of the enzyme was examined after homogenization of cerebral cortices by means which disintegrate nerve endings. In isotonic KCl the enzyme was recovered in solution. In more dilute KCl the enzyme was adsorbed progressively but reversibly to particles, especially at low pH. The subcellular localizations of the enzyme and of acetylcholine formed from choline-14C in vivo were examined further after gentle homogenization of cortices. Subcellular fractions were prepared in density gradients and were sedimented in isotonic NaCl to desorb transferase from microsomes. From isotonic homogenates, 56% of the total enzyme and 60% of acetylcholine-14C were found in nerve endings. From hypotonic homogenates (in which nerve endings are lysed) the enzyme was recovered in solution, whereas 60% of the acetylcholine-14C was found with microsomes. 3H-Acetylcholine added to the homogenizing medium remained in solution. It is concluded that acetylcholine is synthesized in the cytoplasm and is then incorporated into synaptic vesicles. The enzyme was purified from whole brains to a specific activity (Vmax) of 0.727 µmole of product min-1 mg of protein-1 at 38°. The final preparation was free of deacylases, acetylcholinesterase, and carnitine acetyltransferase. By gel filtration the molecular weight of the enzyme was about 50,000; other studies indicated that the enzyme possesses essential —SH groups, that the protein is quite cationic, and that it is soluble in 0.1 mm buffers. Fifteen salts appeared to activate the soluble enzyme. Michaelis constants for the forward reaction were determined; each substrate reduced the affinity for the other substrate. The enzyme-catalyzed reaction was reversible with an apparent equilibrium constant of 514. It is possible that the amount of acetylcholine synthesized in the cytoplasm of nerve endings is regulated by mass action.