naphthylvinylpyridine and homocholine

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.

Topics: Animals; Brain; Choline; Choline O-Acetyltransferase; Hippocampus; Hydrogen-Ion Concentration; Male; Mice; Mice, Inbred ICR; Naphthylvinylpyridine; Rats; Septum Pellucidum; Solubility; Synaptosomes; Tissue Distribution

The choline analog homocholine is not acetylated in vitro by choline-O-acetyltransferase (ChAT, EC 2.3.1.6), which is solubilized by 100 mM-sodium phosphate buffer washes of a crude vesicular fraction of mouse forebrain. However, both homocholine and choline are acetylated by a form of ChAT which is nonionically associated with a subcellular fraction of mouse forebrain containing membrane-associated organelles and occluded acetylcholine (P4). Acetylation of homocholine by membrane-associated ChAT is saturable. 4-(1-Naphthylvinyl)pyridine (NVP) inhibits the acetylation of both choline (60%) and homocholine (40%) by membrane-associated ChAT but reduces the acetylation of choline alone by soluble ChAT (76%). Choline and homocholine serve as competitive alternative substrates for the same membrane-associated ChAT, whereas homocholine acts only as a competitive inhibitor of choline acetylation by soluble ChAT. Acetylhomocholine competitively inhibits the acetylation of choline by both soluble and membrane-associated ChAT more dramatically than does the natural end product, acetylcholine.

Topics: Acetylation; Animals; Brain; Choline; Choline O-Acetyltransferase; Kinetics; Male; Mice; Naphthylvinylpyridine; Neurons; Substrate Specificity