Date of Award
Master of Science in Pharmacology and Toxicology
Pharmacology and Toxicology
Mouse brain choline-0-acetyltransferase (E.C. 22.214.171.124) (ChAT) activity was studied in vitro using a lOOmM sodium phosphate buffer (pH 7.4) wash of a crude vesicular fraction containing solubilized ChAT and a washed crude vesicular fraction containing membrane bound ChAT. Both the solubilized and membrane bound forms of ChAT can acetylate choline linearly for 30 minutes. High concentrations of acetylcholine (Ach) can inhibit solubilized ChAT to a greater degree than the membrane bound enzyme form. FoFtY percent of the Ach synthesized by membrane bound ChAT survives hydrolysis in the presence of an excess of acetylcholinesterase (AchE), whereas none of the Ach synthesized by the solubilized enzyme form does. 4-(l-naphthylvinyl) pyridine inhibits solubilized ChAT more than the membrane bound form, either in vivo or in vitro, and reduces mouse locomotor activity by 80-90%. for 3 hours. Solubilized ChAT is totally sodium dependent whereas the membrane bound form is only partially sodium dependent. Membrane bound ChAT has ·both a high (Km 3.2 uM) and a low affinity (Km=0.48 mM) Michaelis constants as a function of added choline when velocity values are not corrected for acetylation of endogenous choline. Upon correction, only a low affinity Michaelis constant (Km=0.47 mM) is obtained for the membrane bound enzyme form, similar to that of solubilized ChAT (Km=.17 mM). The choline analogues triethylcholine and homocholine are acetylated preferentially by membrane bound ChAT. The results suggest the existence of a membrane bound form of ChAT in mouse brain which is able to synthesize Ach.
Smith, Craig Paul, "A COMPARISON OF SOLUBILIZED AND MEMBRANE BOUND FORMS OF CHOLINE-O-ACETYLTRANSFERASE IN MOUSE BRAIN NERVE ENDINGS" (1979). Open Access Master's Theses. Paper 226.